Oxazole derivatives for use in the treatment of cancer

ABSTRACT

A first aspect of the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt or ester thereof, • B is a saturated or partially unsaturated monocyclic or bicyclic, heterocyclic group optionally substituted by one or more R 10 groups; *X is selected from SO 2 , CO 2 , CO, CONR 11  and (CR 12 R 13 ) p. Said compounds are capable of inhibiting PAICS and are useful in the treatment of proliferative disorders. Further aspects relate to pharmaceutical compositions, therapeutic uses and process for preparing compounds of formula (I).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 filing of InternationalApplication No. PCT/GB2017/053821, filed on Dec. 19, 2017, which claimspriority to Great Britain patent application number 1622362.0, filedDec. 29, 2016.

FIELD OF THE INVENTION

The present invention relates to substituted oxazole derivatives thatare capable of inhibiting PAICS. The compounds find applications in thetreatment of a variety of disorders, including proliferative disorderssuch as cancer.

BACKGROUND TO THE INVENTION

PAICS (phosphorlbosylaminoimidazole carboxylase,phosphoribosylaminoimidazole succinocarboxamide synthetase) is abifunctional 46 kD enzyme catalysing the 6th and 7th steps of the denovo purine pathway (see FIG. 1).

PAICS converts 5-aminoimidazole ribonucleotide (AIR) to4-carboxy-5-aminoimidazole ribonucleotide (CAIR) in an ATP dependentreaction, before finally generating4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide (SAICAR) in acarboxylation reaction (see FIG. 2). This reaction series feeds into theoverall generation of inositiol monophosphate (IMP), a nucleotide thatforms the substrate for AMP and GMP production, from phosphoribosylpyrophosphate (PRPP). The inhibition of folic acid, pyrimidine andpurine biosynthetic pathways has proved an attractive drug target forcancer chemotherapy as rapidly dividing cancer cells have a highbiosynthetic requirement in comparison to non-transformed cells.

Recent literature has highlighted PAICS as an emerging novel target forcancer therapeutics. PAICS was identified as an anti-apoptotic oncogene,with PAICS shRNA protein knock-down reducing the proliferation of amelanoma cell line in vitro. Furthermore, subcutaneous injection ofPAICS knock-down cells in a xenograft model significantly reduced therate of tumour growth (Eilmann et al., PLoS One, 2013 May 22;8(5):e64873).

PAICS expression is significantly upregulated in lung cancer, andmoreover expression levels were related to the prognosis of the patientpopulation; increased expression of PAICS was coupled with tumours of amore aggressive nature. Xenograft models performed using lung cancerPAICS knock-down cells led to a significant reduction in tumour volumeand weight after several weeks (Goswami et al., Oncotarget, 2015 Sep.15; 6(27):23445-61). PAICS over-expression has been also associated witha wide range of other tumour types.

Further studies indicated that PAICS may be a useful biomarker too forpoor prognosis prostate cancer, with heightened expression found inprostate cancer and the severe castration-resistant form, relative tobenign prostate hyperplasia samples (Barfeld et al., Oncotarget, 2015May 20; 6(14):12587-602).

During the recent emergence of PAICS as a potential target for cancertherapeutics, studies have demonstrated that the PAICS gene isoverexpressed as part of a nine gene-expression signature that isstrongly associated with poor-prognosis in triple negative breast cancer(TNBC) patients. Experimental knock-down of any one of these genes had amarked inhibitory effect on cancer cell growth and metastasis in vitroand in vivo. Specifically, shRNA inhibition of PAICS expression stronglyimpaired primary tumour growth when breast cancer cells were injectedorthotopically in the mammary fat pad of mice. Down regulation of PAICSexpression in highly metastatic human breast cancer cells abolished theability of these cells to form metastases to the lungs when injectedintravenously into immunocompromised mice. Of note, this highlypredictive gene-expression signature has a similar prognostic power inbreast cancer patients as the gene-expression signatures, MammaPrint® orOncotypeDX®, currently used in the clinic.

The present invention seeks to provide small molecule inhibitors ofPAICS. In a preferred aspect, the invention seeks to provide smallmolecule inhibitors of PAICS that target the SAICAR synthetase domain.Such small molecule inhibitors have potential therapeutic applicationsin the treatment of proliferative disorders such as cancer.

STATEMENT OF INVENTION

A first aspect of the invention relates to a compound of formula (I), ora pharmaceutically acceptable salt or ester thereof,

wherein:

B is a saturated or partially unsaturated monocyclic or bicyclic,heterocyclic group optionally substituted by one or more R¹⁰ groups andoptionally containing one or more CO groups;

X is selected from SO₂, CO₂, CO, CONR¹¹, NR¹¹CO and (CR¹²R¹³)_(p);

each R¹ is independently selected from halogen, OR¹⁴, SR¹⁴ and R¹⁴;

R² is selected from H and alkyl;

R³ is selected from alkyl, cycloalkyl and heterocycloalkyl, each ofwhich is optionally substituted by one or more substituents selectedfrom NR²⁴R²⁵ and R²⁶; or

R² and R³ are linked together with the nitrogen to which they areattached to form a saturated heterocyclic group optionally containingone or more additional heteroatoms selected from O, N and S, andoptionally substituted by one or more R²⁷ groups;

each R⁴ and R⁵ is independently selected from H, alkyl, (CH₂)_(s)OR¹⁵and (CH₂)_(t)NR¹⁶R¹⁷; or

one of R⁴ and R⁵ is H or alkyl and the other is linked to R³ to form asaturated heterocyclic group;

each R⁶ and R⁷ is independently selected from H, alkyl, (CH₂)_(w)OR¹⁸and (CH₂)_(v)NR¹⁹R²⁰; or

one of R⁶ and R⁷ is H or alkyl and the other is linked to R³ to form asaturated heterocyclic group;

each R⁸ and R⁹ is independently selected from H, alkyl, (CH₂)_(w)OR²¹and (CH₂)_(x)NR²²R²³; or

one of R⁸ and R⁹ is H or alkyl and the other is linked to R³ to form asaturated heterocyclic group; or

one of R⁸ and R⁹ is linked to one of R⁴ and R⁶ to form a cyclic group;

R¹⁰ is selected from alkyl, OH, halogen, alkoxy, CO₂-alkyl, COOH,CO-alkyl and CN;

R¹¹, R¹², R¹³, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴ and R²⁵are each independently H or alkyl;

R¹⁴, R²⁶ and R² are each independently alkyl;

m, q and r are each independently 0, 1 or 2, such that the sum of m+q+ris 2, 3 or 4;

n is an integer selected from 1, 2, 3 and 4;

p is an integer selected from 0, 1 and 2; and

s, t, u, v, w and x are each independently 0, 1, 2, 3 or 4.

A second aspect of the invention relates to a pharmaceutical compositioncomprising at least one compound as described above and apharmaceutically acceptable carrier, diluent or excipient.

A third aspect of the invention relates to a compound as described abovefor use in medicine.

A fourth aspect of the invention relates to a compound as describedabove for use in treating a proliferative disorder.

A fifth aspect of the invention relates to the use of a compound asdescribed above in the preparation of a medicament for treating orpreventing a proliferative disorder.

A sixth aspect of the invention relates to a method of treating aproliferative disorder in a subject in need thereof, said methodcomprising administering to the subject a therapeutically effectiveamount of a compound as described above.

A seventh aspect of the invention relates to a method of treating asubject having a disease state alleviated by inhibition of PAICS,wherein the method comprises administering to the subject atherapeutically effective amount of a compound as described above.

An eighth aspect of the invention relates to the use of a compound asdescribed above in an assay for identifying further candidate compoundscapable of inhibiting PAICS.

A ninth aspect of the invention relates to a combination comprising acompound as described above and a second therapeutic agent.

A tenth aspect of the invention relates to a process for preparingcompounds as described herein.

DETAILED DESCRIPTION

The present invention relates to substituted oxazole derivatives thatare capable of inhibiting PAICS.

“Alkyl” is defined herein as a straight-chain or branched alkyl radical,for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tert-butyl, pentyl, hexyl. Preferably, the alkyl group is a C₁₋₁₂-alkylgroup, more preferably, a C₁₋₆-alkyl group, even more preferably aC₁₋₄-alkyl group.

“Cycloalkyl” is defined herein as a monocyclic alkyl ring, such as,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, or afused bicyclic ring system such as norbornane. Preferably, thecycloalkyl group is a C₃₋₈-cycloalkyl group, more preferably aC₃₋₆-cycloalkyl group.

“Halogen” is defined herein as chloro, fluoro, bromo or iodo.

The compounds of the invention comprise group B, which is a saturated orpartially unsaturated monocyclic or bicyclic heterocyclic group.

As used herein, the term “heterocyclic group” is defined herein as acyclic aliphatic group comprising one or more heteroatoms (that may bethe same or different), such as oxygen, nitrogen or sulfur, which isoptionally interrupted by one or more —(CO)— groups in the ring.Preferably, the heterocyclic group is a C₃-C₇-heterocycloalkyl group,more preferably a C₃-C₆-heterocycloalkyl group. Alternatively, theheterocycloalkyl group is a C₄-C₇-heterocycloalkyl, more preferably aC₄-C₆-heterocycloalkyl.

Preferably, the heterocyclic group B is saturated. However, in certainembodiments, the heterocyclic group may contain one or more double bondssuch that it is partially unsaturated.

Preferably, the heterocyclic group B is a monocylic saturatedheterocyclic group. Particularly preferred monocylic saturatedheterocyclic groups include, but are not limited to, piperazinyl,piperidinyl, morpholinyl, thiomorpholinyl, pyrrolidinyl,tetrahydrofuranyl and tetrahydropyranyl.

More preferably, heterocyclic group B is selected from piperazinyl,piperidinyl, morpholinyl, thiomorpholinyl and pyrrolidinyl. Morepreferably still, heterocyclic group B is selected from piperazinyl,piperidinyl and pyrrolidinyl.

In one preferred embodiment, heterocyclic group B is substituted by oneor more R¹⁰ groups, preferably by one R¹⁰ group. In another preferredembodiment, heterocyclic group B is unsubstituted.

In one preferred embodiment, heterocyclic group B contains one or moreCO groups, preferably one CO group.

Preferably, where:

-   -   one of R⁶ and R⁷ is H or alkyl (more preferably methyl) and the        other is linked to R³ to form a saturated heterocyclic group, or    -   one of R⁸ and R⁹ is H or alkyl (more preferably methyl) and the        other is linked to R³ to form a saturated heterocyclic group, or    -   one of R⁴ and R⁵ is H or alkyl (more preferably methyl) and the        other is linked to R³ to form a saturated heterocylic group;

the saturated heterocyclic group is a 4-, 5- or 6-membered heterocyclicgroup, more preferably, 5- or 6-membered heterocyclic group, even morepreferably, a pyrrolidinyl or piperidinyl group.

In one preferred embodiment of the invention, B is a monocyclic 5- or6-membered saturated or partially unsaturated heterocyclic groupoptionally containing one or more CO groups and optionally substitutedby one or more R¹⁰ groups.

In one preferred embodiment of the invention, B is a monocyclic 5- or6-membered saturated or partially unsaturated heterocyclic groupoptionally substituted by one or more R¹⁰ groups.

In one particularly preferred embodiment, B is a piperazinyl group or apiperidinyl group, each of which is optionally substituted with one ormore R¹⁰ groups.

In another particularly preferred embodiment, B is a piperazinyl groupor a piperidinyl group, wherein for each group, one of the methylenegroups is replaced with a CO group.

In one preferred embodiment of the invention:

each R⁴ and R⁵ is independently selected from H and alkyl; or

one of R⁴ and R⁵ is H or alkyl and the other is linked to R³ to form asaturated heterocyclic group.

In one preferred embodiment of the invention:

each R⁸ and R⁷ is independently selected from H, alkyl, (CH₂)_(u)OR¹⁸and (CH₂)NR¹⁹R²⁰; or one of R⁶ and R⁷ is H or alkyl and the other islinked to R³ to form a saturated heterocyclic group.

In one preferred embodiment of the invention:

each R⁸ and R⁹ is independently selected from H, alkyl, (CH₂)_(w)OR²¹and (CH₂)_(x)NR²²R²³; or one of R⁸ and R⁹ is H or alkyl and the other islinked to R³ to form a saturated heterocyclic group.

In one preferred embodiment of the invention:

each R⁴ and R⁵ is independently selected from H and alkyl; or

one of R⁴ and R⁵ is H or alkyl and the other is linked to R³ to form asaturated heterocyclic group;

each R⁶ and R⁷ is independently selected from H, alkyl, (CH₂)_(u)OR¹⁸and (CH₂)_(v)NR¹⁹R²⁰; or one of R⁵ and R⁷ is H or alkyl and the other islinked to R³ to form a saturated heterocyclic group; and

each R⁸ and R⁹ is independently selected from H, alkyl, (CH₂)_(w)OR²¹and (CH₂)_(x)NR²²R²³; or one of R⁸ and R⁹ is H or alkyl and the other islinked to R³ to form a saturated heterocyclic group.

In one preferred embodiment of the invention, the compound is of formula(Ib), or a pharmaceutically acceptable salt or ester thereof,

wherein Y and Z are both N, or one of Y and Z is N, and the other is CH;and

R¹⁻⁹, X, m, q and r are as defined above.

In one preferred embodiment, Y and Z are both N.

In one preferred embodiment, R¹ is selected from Br, I, Cl, OMe, SMe andMe. More preferably, R¹ is selected from Br, Cl and SMe.

In one preferred embodiment, X is CO₂ or SO₂, more preferably SO₂.

In one preferred embodiment, R² is selected from H, methyl, ethyl andisopropyl; and

R³ is selected from methyl, ethyl, isopropyl and cyclopropyl.

In one preferred embodiment, R² and R³ are linked together with thenitrogen to which they are attached to form a 5- or 6-membered saturatedheterocyclic group, preferably a pyrrolidinyl group.

In one preferred embodiment:

m is 1 or 2;

q is 1 or 2, more preferably 1;

r is 0;

R⁸ and R⁹ are each independently H or alkyl, more preferably H; and

R⁶ and R⁷ are each independently H or alkyl, more preferably H.

In another preferred embodiment:

m is 1;

q is 1 or 2;

r is 0;

one of R⁸ and R⁹ is H or alkyl and the other is linked to R³ to form asaturated heterocyclic group; and

each R⁶ and R⁷ is independently H or alkyl.

In another preferred embodiment:

m is 1;

q is 1;

r is 1 or 2, preferably 1;

R⁸ and R⁹ are each independently H or alkyl, more preferably H;

one of R⁶ and R⁷ is H or alkyl and the other is linked to R³ to form asaturated heterocyclic group; and

R⁴ and R⁵ are each independently H or alkyl, more preferably H.

In one highly preferred embodiment, the compound of the invention isselected from compounds shown in the table below:

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

44

45

46

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

84

85

86

87

88

89

and pharmaceutically acceptable salts and esters thereof.

In one even more highly preferred embodiment, the compound is selectedfrom 21, 23, 24, 25, 54, 55, 67, 69 and 70.

Therapeutic Applications

A further aspect of the invention relates to a compound as describedabove for use in medicine.

Another aspect of the invention relates to a compound as described abovefor use in treating a proliferative disorder.

The term “proliferative disorder” is used herein in a broad sense toinclude any disorder that requires control of the cell cycle, forexample cardiovascular disorders such as restenosis and cardiomyopathy,auto-immune disorders such as glomerulonephritis and rheumatoidarthritis, dermatological disorders such as psoriasis,anti-inflammatory, anti-fungal, antiparasitic disorders such as malaria,emphysema and alopecia. In these disorders, the compounds of the presentinvention may induce apoptosis or maintain stasis within the desiredcells as required.

In one preferred embodiment, the invention relates to a compound asdescribed above for use in preventing or reducing metastasis. Thus, inone preferred embodiment, the compound is for use in preventing oralleviating or treating metastatic cancer, for example, secondarymalignant growths at a distance from a primary site of cancer.

In another preferred embodiment, the invention relates to a compound asdescribed above for use in blocking cell growth.

In one preferred embodiment, the proliferative disorder is cancer orleukemia. Preferably, the cancer is selected from solid cancers at anystage. In another preferred embodiment, the cancer is in a late-stage,with metastatic lesions.

Preferably, the cancer is selected from breast cancer, colon cancer,prostate melanoma, bladder, pancreatic, head and neck and ovariancancer, with or without metastasis, and haematological cancers such asacute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acutelymphocytic leukemia (ALL), multiple myeloma (MM) and non-Hodgkinslymphoma.

In one preferred embodiment, the proliferative disorder is selected frombreast cancer, colon cancer, lung cancer, melanoma and prostate cancer.Studies by the applicant have demonstrated that PAICS mRNA isupregulated in these tumour types.

In one particularly preferred embodiment, the proliferative disorder isbreast cancer. More preferably, the proliferative disorder is metastaticbreast cancer or triple negative breast cancer (TNBC). Triple-negativebreast cancer refers to any breast cancer that does not express thegenes for estrogen receptor (ER), progesterone receptor (PR) orHer2/neu. This makes it more difficult to treat since mostchemotherapies target one of the three receptors, so triple-negativecancers often require combination therapies.

Another aspect relates to the use of a compound as described above inthe preparation of a medicament for treating or preventing aproliferative disorder, for example, cancer or leukemia.

Another aspect relates to method of treating a proliferative disorder ina subject in need thereof, said method comprising administering to thesubject a therapeutically effective amount of a compound as describedabove.

Preferably, the compound is administered in an amount sufficient toinhibit PAICS.

Another aspect relates to a compound of the invention for use in theprevention or treatment of a disorder caused by, associated with oraccompanied by any abnormal activity against a biological target,wherein the target is PAICS.

Yet another aspect relates to the use of a compound of the invention inthe preparation of a medicament for the prevention or treatment of adisorder caused by, associated with or accompanied by any abnormalactivity against a biological target, wherein the target is PAICS.

Another aspect of the invention relates to a method of treating a PAICSrelated disease or disorder. The method according to this aspect of thepresent invention is effected by administering to a subject in needthereof a therapeutically effective amount of a compound of the presentinvention, as described hereinabove, either per se, or, more preferably,as a part of a pharmaceutical composition, mixed with, for example, apharmaceutically acceptable carrier, as is detailed hereinafter.

Yet another aspect of the invention relates to a method of treating amammal having a disease state alleviated by inhibition of PAICS, whereinthe method comprises administering to a mammal a therapeuticallyeffective amount of a compound according to the invention.

Preferably, the subject is a mammal, more preferably a human.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of the chemical, pharmacological, biological, biochemicaland medical arts.

The term “administering” as used herein refers to a method for bringinga compound of the present invention and PAICS together in such a mannerthat the compound can affect the enzyme activity of the PAICS eitherdirectly; i.e., by interacting with the PAICS itself or indirectly;i.e., by interacting with another molecule on which the catalyticactivity of the PAICS is dependent. As used herein, administration canbe accomplished either in vitro, i.e. in a test tube, or in vivo, i.e.,in cells or tissues of a living organism.

Herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease ordisorder, substantially ameliorating clinical symptoms of a disease ordisorder or substantially preventing the appearance of clinical symptomsof a disease or disorder.

Herein, the term “preventing” refers to a method for barring an organismfrom acquiring a disorder or disease in the first place.

The term “therapeutically effective amount” refers to that amount of thecompound being administered which will relieve to some extent one ormore of the symptoms of the disease or disorder being treated.

For any compound used in this invention, a therapeutically effectiveamount, also referred to herein as a therapeutically effective dose, canbe estimated initially from cell culture assays. For example, a dose canbe formulated in animal models to achieve a circulating concentrationrange that includes the IC₅₀ or the IC₁₀₀ as determined in cell culture.Such information can be used to more accurately determine useful dosesin humans. Initial dosages can also be estimated from in vivo data.Using these initial guidelines one of ordinary skill in the art coulddetermine an effective dosage in humans.

Moreover, toxicity and therapeutic efficacy of the compounds describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the LD₅₀ and theED₅₀. The dose ratio between toxic and therapeutic effect is thetherapeutic index and can be expressed as the ratio between LD₅₀ andED₅₀. Compounds which exhibit high therapeutic indices are preferred.The data obtained from these cell cultures assays and animal studies canbe used in formulating a dosage range that is not toxic for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (see, e.g.,Fingl et al, 1975, In: The Pharmacological Basis of Therapeutics,chapter 1, page 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active compound which are sufficient to maintaintherapeutic effect. Usual patient dosages for oral administration rangefrom about 50-2000 mg/kg/day, commonly from about 100-1000 mg/kg/day,preferably from about 150-700 mg/kg/day and most preferably from about250-500 mg/kg/day. Preferably, therapeutically effective serum levelswill be achieved by administering multiple doses each day. In cases oflocal administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.One skilled in the art will be able to optimize therapeuticallyeffective local dosages without undue experimentation.

As used herein, “PAICS related disease or disorder” refers to a diseaseor disorder characterized by inappropriate or abnormal PAICS activity orover-activity. Inappropriate or abnormal activity refers to either; (i)expression in cells which normally do not express the protein; (ii)increased expression leading to unwanted cell proliferation,differentiation and/or growth; or, (iii) decreased expression leading tounwanted reductions in cell proliferation, differentiation and/orgrowth. Over-activity of PAICS refers to either amplification of thegene encoding PAICS or production of a level of PAICS activity, whichcan correlate with a cell proliferation, differentiation and/or growthdisorder (that is, as the level of the PAICS increases, the severity ofone or more of the symptoms of the cellular disorder increases).Over-activity can also be the result of ligand-independent orconstitutive activation as a result of mutations such as deletions of afragment of the protein responsible for ligand binding.

Thus, the present invention further provides use of compounds as definedherein for the manufacture of medicaments for the treatment of diseaseswhere it is desirable to inhibit PAICS. Such diseases includeproliferative disorders such as cancer or leukemia.

Pharmaceutical Compositions

For use according to the present invention, the compounds orphysiologically acceptable salt, ester or other physiologicallyfunctional derivative thereof, described herein, may be presented as apharmaceutical formulation, comprising the compounds or physiologicallyacceptable salt, ester or other physiologically functional derivativethereof, together with one or more pharmaceutically acceptable carriersand optionally other therapeutic and/or prophylactic ingredients. Thecarrier(s) must be acceptable in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient thereof. The pharmaceutical compositions may be for human oranimal usage in human and veterinary medicine.

Examples of such suitable excipients for the various different forms ofpharmaceutical compositions described herein may be found in theHandbook of Pharmaceutical Excipients, 2^(nd) Edition, (1994), Edited byA Wade and P J Weller.

Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).

Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol and the like. Examplesof suitable diluents include ethanol, glycerol and water.

The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as, or in addition to, the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s), buffer(s), flavouring agent(s), surface activeagent(s), thickener(s), preservative(s) (including anti-oxidants) andthe like, and substances included for the purpose of rendering theformulation isotonic with the blood of the intended recipient.

Examples of suitable binders include starch, gelatin, natural sugarssuch as glucose, anhydrous lactose, free-flow lactose, beta-lactose,corn sweeteners, natural and synthetic gums, such as acacia, tragacanthor sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavoring agents maybe provided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

Pharmaceutical formulations include those suitable for oral, topical(including dermal, buccal and sublingual), rectal or parenteral(including subcutaneous, intradermal, intramuscular and intravenous),nasal and pulmonary administration e.g., by inhalation. The formulationmay, where appropriate, be conveniently presented in discrete dosageunits and may be prepared by any of the methods well known in the art ofpharmacy.

All methods include the step of bringing into association an activecompound with liquid carriers or finely divided solid carriers or bothand then, if necessary, shaping the product into the desiredformulation.

Pharmaceutical formulations suitable for oral administration wherein thecarrier is a solid are most preferably presented as unit doseformulations such as boluses, capsules or tablets each containing apredetermined amount of active compound. A tablet may be made bycompression or moulding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine an active compound in a free-flowing form such as apowder or granules optionally mixed with a binder, lubricant, inertdiluent, lubricating agent, surface-active agent or dispersing agent.Moulded tablets may be made by moulding an active compound with an inertliquid diluent. Tablets may be optionally coated and, if uncoated, mayoptionally be scored. Capsules may be prepared by filling an activecompound, either alone or in admixture with one or more accessoryingredients, into the capsule shells and then sealing them in the usualmanner. Cachets are analogous to capsules wherein an active compoundtogether with any accessory ingredient(s) is sealed in a rice paperenvelope. An active compound may also be formulated as dispersiblegranules, which may for example be suspended in water beforeadministration, or sprinkled on food. The granules may be packaged,e.g., in a sachet. Formulations suitable for oral administration whereinthe carrier is a liquid may be presented as a solution or a suspensionin an aqueous or non-aqueous liquid, or as an oil-in-water liquidemulsion.

Formulations for oral administration include controlled release dosageforms, e.g., tablets wherein an active compound is formulated in anappropriate release-controlling matrix, or is coated with a suitablerelease-controlling film. Such formulations may be particularlyconvenient for prophylactic use.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art. The suppositories may beconveniently formed by admixture of an active compound with the softenedor melted carrier(s) followed by chilling and shaping in moulds.Pharmaceutical formulations suitable for parenteral administrationinclude sterile solutions or suspensions of an active compound inaqueous or oleaginous vehicles.

Injectable preparations may be adapted for bolus injection or continuousinfusion. Such preparations are conveniently presented in unit dose ormulti-dose containers which are sealed after introduction of theformulation until required for use. Alternatively, an active compoundmay be in powder form which is constituted with a suitable vehicle, suchas sterile, pyrogen-free water, before use.

An active compound may also be formulated as long-acting depotpreparations, which may be administered by intramuscular injection or byimplantation, e.g., subcutaneously or intramuscularly. Depotpreparations may include, for example, suitable polymeric or hydrophobicmaterials, or ion-exchange resins. Such long-acting formulations areparticularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the buccal cavityare presented such that particles containing an active compound anddesirably having a diameter in the range of 0.5 to 7 microns aredelivered in the bronchial tree of the recipient.

As one possibility such formulations are in the form of finelycomminuted powders which may conveniently be presented either in apierceable capsule, suitably of, for example, gelatin, for use in aninhalation device, or alternatively as a self-propelling formulationcomprising an active compound, a suitable liquid or gaseous propellantand optionally other ingredients such as a surfactant and/or a soliddiluent. Suitable liquid propellants include propane and thechlorofluorocarbons, and suitable gaseous propellants include carbondioxide. Self-propelling formulations may also be employed wherein anactive compound is dispensed in the form of droplets of solution orsuspension.

Such self-propelling formulations are analogous to those known in theart and may be prepared by established procedures. Suitably they arepresented in a container provided with either a manually-operable orautomatically functioning valve having the desired spraycharacteristics; advantageously the valve is of a metered typedelivering a fixed volume, for example, 25 to 100 microlitres, upon eachoperation thereof.

As a further possibility an active compound may be in the form of asolution or suspension for use in an atomizer or nebuliser whereby anaccelerated airstream or ultrasonic agitation is employed to produce afine droplet mist for inhalation.

Formulations suitable for nasal administration include preparationsgenerally similar to those described above for pulmonary administration.When dispensed such formulations should desirably have a particlediameter in the range 10 to 200 microns to enable retention in the nasalcavity; this may be achieved by, as appropriate, use of a powder of asuitable particle size or choice of an appropriate valve. Other suitableformulations include coarse powders having a particle diameter in therange 20 to 500 microns, for administration by rapid inhalation throughthe nasal passage from a container held close up to the nose, and nasaldrops comprising 0.2 to 5% w/v of an active compound in aqueous or oilysolution or suspension.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Additionally, such pharmaceuticallyacceptable carriers may be aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Preservatives and other additives mayalso be present, such as, for example, antimicrobials, antioxidants,chelating agents, inert gases and the like.

Formulations suitable for topical formulation may be provided forexample as gels, creams or ointments. Such preparations may be appliede.g. to a wound or ulcer either directly spread upon the surface of thewound or ulcer or carried on a suitable support such as a bandage,gauze, mesh or the like which may be applied to and over the area to betreated.

Liquid or powder formulations may also be provided which can be sprayedor sprinkled directly onto the site to be treated, e.g. a wound orulcer. Alternatively, a carrier such as a bandage, gauze, mesh or thelike can be sprayed or sprinkled with the formulation and then appliedto the site to be treated.

According to a further aspect of the invention, there is provided aprocess for the preparation of a pharmaceutical or veterinarycomposition as described above, the process comprising bringing theactive compound(s) into association with the carrier, for example byadmixture.

In general, the formulations are prepared by uniformly and intimatelybringing into association the active agent with liquid carriers orfinely divided solid carriers or both, and then if necessary shaping theproduct. The invention extends to methods for preparing a pharmaceuticalcomposition comprising bringing a compound of general formula (I) inconjunction or association with a pharmaceutically or veterinarilyacceptable carrier or vehicle.

Salts/Esters

The compounds of the invention can be present as salts or esters, inparticular pharmaceutically and veterinarily acceptable salts or esters.

Pharmaceutically acceptable salts of the compounds of the inventioninclude suitable acid addition or base salts thereof. A review ofsuitable pharmaceutical salts may be found in Berge et al, J Pharm Sci,66, 1-19 (1977). Salts are formed, for example with strong inorganicacids such as mineral acids, e.g. hydrohalic acids such ashydrochloride, hydrobromide and hydroiodide, sulfuric acid, phosphoricacid sulfate, bisulfate, hemisulfate, thiocyanate, persulfate andsulfonic acids; with strong organic carboxylic acids, such asalkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted orsubstituted (e.g., by halogen), such as acetic acid; with saturated orunsaturated dicarboxylic acids, for example oxalic, malonic, succinic,maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylicacids, for example ascorbic, glycolic, lactic, malic, tartaric or citricacid; with amino acids, for example aspartic or glutamic acid; withbenzoic acid; or with organic sulfonic acids, such as (C₁-C₄)-alkyl- oraryl-sulfonic acids which are unsubstituted or substituted (for example,by a halogen) such as methane- or p-toluene sulfonic acid. Salts whichare not pharmaceutically or veterinarily acceptable may still bevaluable as intermediates.

Preferred salts include, for example, acetate, trifluoroacetate,lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate,adipate, alginate, aspartate, benzoate, butyrate, digluconate,cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate,hexanoate, fumarate, nicotinate, palmoate, pectinate,3-phenylpropionate, picrate, pivalate, proprionate, tartrate,lactobionate, pivolate, camphorate, undecanoate and succinate; organicsulfonic acids such as methanesulfonate, ethanesulfonate,2-hydroxyethane sulfonate, camphorsulfonate, 2-naphthalenesulfonate,benzenesulfonate, p-chlorobenzenesulfonate and p-toluenesulfonate; andinorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulfate, bisulfate, hemisulfate, thiocyanate, persulfate, phosphoric andsulfonic acids.

Esters are formed either using organic acids or alcohols/hydroxides,depending on the functional group being esterified. Organic acidsinclude carboxylic acids, such as alkanecarboxylic acids of 1 to 12carbon atoms which are unsubstituted or substituted (e.g., by halogen),such as acetic acid; with saturated or unsaturated dicarboxylic acid,for example oxalic, malonic, succinic, maleic, fumaric, phthalic ortetraphthalic; with hydroxycarboxylic acids, for example ascorbic,glycolic, lactic, malic, tartaric or citric acid; with amino acids, forexample aspartic or glutamic acid; with benzoic acid; or with organicsulfonic acids, such as (C₁-C₄)-alkyl- or aryl-sulfonic acids which areunsubstituted or substituted (for example, by a halogen) such asmethane- or p-toluene sulfonic acid. Suitable hydroxides includeinorganic hydroxides, such as sodium hydroxide, potassium hydroxide,calcium hydroxide, aluminium hydroxide. Alcohols include alkane alcoholsof 1-12 carbon atoms which may be unsubstituted or substituted, (e.g. bya halogen).

Enantiomers/Tautomers

In all aspects of the present invention previously discussed, theinvention includes, where appropriate all enantiomers, diastereoisomersand tautomers of the compounds of the invention. The person skilled inthe art will recognise compounds that possess optical properties (one ormore chiral carbon atoms) or tautomeric characteristics. Thecorresponding enantiomers and/or tautomers may be isolated/prepared bymethods known in the art.

Enantiomers are characterised by the absolute configuration of theirchiral centres and described by the R- and S-sequencing rules of Cahn,Ingold and Prelog. Such conventions are well known in the art (e.g. see‘Advanced Organic Chemistry’, 3^(rd) edition, ed. March, J., John Wileyand Sons, New York, 1985).

Compounds of the invention containing a chiral centre may be used as aracemic mixture, an enantiomerically enriched mixture, or the racemicmixture may be separated using well-known techniques and an individualenantiomer may be used alone.

Stereo and Geometric Isomers

Some of the compounds of the invention may exist as stereoisomers and/orgeometric isomers—e.g. they may possess one or more asymmetric and/orgeometric centres and so may exist in two or more stereoisomeric and/orgeometric forms. The present invention contemplates the use of all theindividual stereoisomers and geometric isomers of those inhibitoragents, and mixtures thereof. The terms used in the claims encompassthese forms, provided said forms retain the appropriate functionalactivity (though not necessarily to the same degree).

Isotopic Variations

The present invention also includes all suitable isotopic variations ofthe agent or a pharmaceutically acceptable salt thereof. An isotopicvariation of an agent of the present invention or a pharmaceuticallyacceptable salt thereof is defined as one in which at least one atom isreplaced by an atom having the same atomic number but an atomic massdifferent from the atomic mass usually found in nature. Examples ofisotopes that can be incorporated into the agent and pharmaceuticallyacceptable salts thereof include isotopes of hydrogen, carbon, nitrogen,oxygen, phosphorus, sulfur, fluorine and chlorine such as ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁶S, ³⁵S, ¹⁸F and ³⁰Cl, respectively.Certain isotopic variations of the agent and pharmaceutically acceptablesalts thereof, for example, those in which a radioactive isotope such as³H or ¹⁴C is incorporated, are useful in drug and/or substrate tissuedistribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C,isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with isotopes such as deuterium,i.e., ²H, may afford certain therapeutic advantages resulting fromgreater metabolic stability, for example, increased in vivo half-life orreduced dosage requirements and hence may be preferred in somecircumstances. For example, the invention includes compounds of generalformula (I) where any hydrogen atom has been replaced by a deuteriumatom. Isotopic variations of the agent of the present invention andpharmaceutically acceptable salts thereof of this invention cangenerally be prepared by conventional procedures using appropriateisotopic variations of suitable reagents.

Prodrugs

The invention further includes the compounds of the present invention inprodrug form, i.e. covalently bonded compounds which release the activeparent drug according to general formula (I) in vivo. Such prodrugs aregenerally compounds of the invention wherein one or more appropriategroups have been modified such that the modification may be reversedupon administration to a human or mammalian subject. Reversion isusually performed by an enzyme naturally present in such subject, thoughit is possible for a second agent to be administered together with sucha prodrug in order to perform the reversion in vivo. Examples of suchmodifications include esters (for example, any of those describedabove), wherein the reversion may be carried out be an esterase etc.Other such systems will be well known to those skilled in the art.

Solvates

The present invention also includes solvate forms of the compounds ofthe present invention. The terms used in the claims encompass theseforms.

Polymorphs

The invention further relates to the compounds of the present inventionin their various crystalline forms, polymorphic forms and (an)hydrousforms. It is well established within the pharmaceutical industry thatchemical compounds may be isolated in any of such forms by slightlyvarying the method of purification and or isolation form the solventsused in the synthetic preparation of such compounds.

Administration

The pharmaceutical compositions of the present invention may be adaptedfor rectal, nasal, intrabronchial, topical (including buccal andsublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intraarterial and intradermal),intraperitoneal or intrathecal administration. Preferably theformulation is an orally administered formulation. The formulations mayconveniently be presented in unit dosage form, i.e., in the form ofdiscrete portions containing a unit dose, or a multiple or sub-unit of aunit dose. By way of example, the formulations may be in the form oftablets and sustained release capsules, and may be prepared by anymethod well known in the art of pharmacy.

Formulations for oral administration in the present invention may bepresented as: discrete units such as capsules, gellules, drops, cachets,pills or tablets each containing a predetermined amount of the activeagent; as a powder or granules; as a solution, emulsion or a suspensionof the active agent in an aqueous liquid or a non-aqueous liquid; or asan oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or asa bolus etc. Preferably, these compositions contain from 1 to 250 mg andmore preferably from 10-100 mg, of active ingredient per dose.

For compositions for oral administration (e.g. tablets and capsules),the term “acceptable carrier” includes vehicles such as commonexcipients e.g. binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose,ethylcellulose, sodium carboxymethylcellulose,hydroxypropyl-methylcellulose, sucrose and starch; fillers and carriers,for example corn starch, gelatin, lactose, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride andalginic acid; and lubricants such as magnesium stearate, sodium stearateand other metallic stearates, glycerol stearate stearic acid, siliconefluid, talc waxes, oils and colloidal silica. Flavouring agents such aspeppermint, oil of wintergreen, cherry flavouring and the like can alsobe used. It may be desirable to add a colouring agent to make the dosageform readily identifiable. Tablets may also be coated by methods wellknown in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may be optionally coated or scored and may be formulated soas to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

Other forms of administration comprise solutions or emulsions which maybe injected intravenously, intraarterially, intrathecally,subcutaneously, intradermally, intraperitoneally or intramuscularly, andwhich are prepared from sterile or sterilisable solutions. Injectableforms typically contain between 10-1000 mg, preferably between 10-250mg, of active ingredient per dose.

The pharmaceutical compositions of the present invention may also be inform of suppositories, pessaries, suspensions, emulsions, lotions,ointments, creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skinpatch. For example, the active ingredient can be incorporated into acream consisting of an aqueous emulsion of polyethylene glycols orliquid paraffin. The active ingredient can also be incorporated, at aconcentration of between 1 and 10% by weight, into an ointmentconsisting of a white wax or white soft paraffin base together with suchstabilisers and preservatives as may be required.

Dosage

A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will depend on a variety of factors includingthe activity of the specific compound employed, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and theindividual undergoing therapy. The dosages disclosed herein areexemplary of the average case. There can of course be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

In accordance with this invention, an effective amount of a compound ofgeneral formula (I) may be administered to inhibit PAICS. Of course,this dosage amount will further be modified according to the type ofadministration of the compound. For example, to achieve an “effectiveamount” for acute therapy, parenteral administration of a compound ofgeneral formula (I) is preferred. An intravenous infusion of thecompound in 5% dextrose in water or normal saline, or a similarformulation with suitable excipients, is most effective, although anintramuscular bolus injection is also useful. Typically, the parenteraldose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and20 mg/kg, in a manner to maintain the concentration of drug in theplasma at a concentration effective to inhibit a kinase. The compoundsmay be administered one to four times daily at a level to achieve atotal daily dose of about 0.4 to about 400 mg/kg/day. The precise amountof an inventive compound which is therapeutically effective, and theroute by which such compound is best administered, is readily determinedby one of ordinary skill in the art by comparing the blood level of theagent to the concentration required to have a therapeutic effect.

The compounds of this invention may also be administered orally to thepatient, in a manner such that the concentration of drug is sufficientto achieve one or more of the therapeutic indications disclosed herein.Typically, a pharmaceutical composition containing the compound isadministered at an oral dose of between about 0.1 to about 50 mg/kg in amanner consistent with the condition of the patient. Preferably the oraldose would be about 0.5 to about 20 mg/kg.

No unacceptable toxicological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention. The compounds of this invention, which may have goodbioavailability, may be tested in one of several biological assays todetermine the concentration of a compound which is required to have agiven pharmacological effect.

Combinations

In a particularly preferred embodiment, the one or more compounds of theinvention are administered in combination with one or more other activeagents, for example, existing drugs available on the market. In suchcases, the compounds of the invention may be administered consecutively,simultaneously or sequentially with the one or more other active agents.

Drugs in general are more effective when used in combination. Inparticular, combination therapy is desirable in order to avoid anoverlap of major toxicities, mechanism of action and resistancemechanism(s). Furthermore, it is also desirable to administer most drugsat their maximum tolerated doses with minimum time intervals betweensuch doses. The major advantages of combining chemotherapeutic drugs arethat it may promote additive or possible synergistic effects throughbiochemical interactions and also may decrease or delay the emergence ofresistance.

Beneficial combinations may be suggested by studying the inhibitoryactivity of the test compounds with agents known or suspected of beingvaluable in the treatment of a particular disorder. For example, theinvention relates to the use of a compound as described above in anassay for identifying compounds that promote additive and synergisticactivity upon anti-cancer activities when combined with the compound.Preferably the assay is a high-throughput cell based phenotypic screen.This procedure can also be used to determine the order of administrationof the agents, i.e. before, simultaneously, or after delivery. Suchscheduling may be a feature of all the active agents identified herein.

Assay

A further aspect of the invention relates to the use of a compound asdescribed above in an assay for identifying further candidate compoundscapable of inhibiting PAICS. Preferably, the candidate compound iscapable of selectively inhibiting PAICS.

Preferably, the assay is a competitive binding assay.

More preferably, the competitive binding assay comprises contacting acompound of the invention with PAICS, and a candidate compound anddetecting any change in the interaction between the compound accordingto the invention and the PAICS. Preferably, the candidate compound isgenerated by conventional SAR modification of a compound of theinvention.

As used herein, the term “conventional SAR modification” refers tostandard methods known in the art for varying a given compound by way ofchemical derivatisation.

Thus, in one aspect, the identified compound may act as a model (forexample, a template) for the development of other compounds. Thecompounds employed in such a test may be free in solution, affixed to asolid support, borne on a cell surface, or located intracellularly. Theabolition of activity or the formation of binding complexes between thecompound and the agent being tested may be measured.

The assay of the present invention may be a screen, whereby a number ofagents are tested. In one aspect, the assay method of the presentinvention is a high throughput screen.

This invention also contemplates the use of competitive drug screeningassays in which neutralising antibodies capable of binding a compoundspecifically compete with a test compound for binding to a compound.

Another technique for screening provides for high throughput screening(HTS) of agents having suitable binding affinity to the substances andis based upon the method described in detail in WO 84/03564.

It is expected that the assay methods of the present invention will besuitable for both small and large-scale screening of test compounds aswell as in quantitative assays.

Preferably, the competitive binding assay comprises contacting acompound of the invention with a kinase in the presence of a knownsubstrate of said kinase and detecting any change in the interactionbetween said kinase and said known substrate.

A further aspect of the invention provides a method of detecting thebinding of a ligand to PAICS, said method comprising the steps of:

-   (i) contacting a ligand with PAICS in the presence of a known    substrate of said kinase;-   (ii) detecting any change in the interaction between said PAICS and    said known substrate;

and wherein said ligand is a compound of the invention.

One aspect of the invention relates to a process comprising the stepsof:

(a) performing an assay method described hereinabove;

(b) identifying one or more ligands capable of binding to a ligandbinding domain; and

(c) preparing a quantity of said one or more ligands.

Another aspect of the invention provides a process comprising the stepsof:

-   (a) performing an assay method described hereinabove;-   (b) identifying one or more ligands capable of binding to a ligand    binding domain; and-   (c) preparing a pharmaceutical composition comprising said one or    more ligands.

Another aspect of the invention provides a process comprising the stepsof:

-   (a) performing an assay method described hereinabove;-   (b) identifying one or more ligands capable of binding to a ligand    binding domain;-   (c) modifying said one or more ligands capable of binding to a    ligand binding domain;-   (d) performing the assay method described hereinabove;-   (e) optionally preparing a pharmaceutical composition comprising    said one or more ligands.

The invention also relates to a ligand identified by the methoddescribed hereinabove.

Yet another aspect of the invention relates to a pharmaceuticalcomposition comprising a ligand identified by the method describedhereinabove.

Another aspect of the invention relates to the use of a ligandidentified by the method described hereinabove in the preparation of apharmaceutical composition for use in the treatment of one or moredisorders as described hereinabove.

The above methods may be used to screen for a ligand useful as aninhibitor of one or more kinases.

Compounds of general formula (I) are useful both as laboratory tools andas therapeutic agents. In the laboratory certain compounds of theinvention are useful in establishing whether a known or newly discoveredprotein contributes a critical or at least significant biochemicalfunction during the establishment or progression of a disease state, aprocess commonly referred to as ‘target validation’.

Synthesis

Another aspect of the invention relates to a process for preparing acompound of formula (Ib) as defined herein, wherein Y and Z are both N,said process comprising the steps of:

(i) preparing an intermediate of formula (II), where R¹ is as definedhereinabove;

(ii) converting said intermediate of formula (II) into a compound offormula (Ib).

The invention is further described by way of the following non-limitingexamples, and with reference to the following figures, wherein:

FIG. 1 shows the domains of phosphoribosylaminoimidazole carboxylase,phosphoribosylaminoimidazole succinocarboxamide synthetase, and its rolein de novo purine biosynthesis.

FIG. 2 shows the reaction catalysed by human PAICS.

FIG. 3 shows the Transcreener FI assay principle.

FIG. 4 shows the effect of Compound 69 in a xenograft model(MDA-MB-231-Dlux Xenograft Model), showing tumour volume against studydays.

FIG. 5 shows the mean tumour weight (mg) measured in the differentexperimental groups (WT, Ctrl 1, KD 3 and KD 14).

FIG. 6 shows representative images of tumours for each group (3 tumoursper group) in the evaluation of the growth of PAICS CRISPR KD MDA231cells in a Chorioallantoic Membrane (CAM) model. PAICS CRISPR KD clones(KD3 and KD14) of MDA231 modified cells showed a significant reductionin tumour development compared to wild type and control modified MDA231cells.

EXAMPLES

Materials and Methods

Chromatography

Preparative high pressure liquid chromatography was carried out usingapparatus made by Agilent. The apparatus is constructed such that thechromatography is monitored by a multi-wavelength UV detector (G1365Bmanufactured by Agilent) and an MM-ES+APCI mass spectrometer (G-1956A,manufactured by Agilent) connected in series, and if the appropriatecriteria are met the sample is collected by an automated fractioncollector (G1364B manufactured by Agilent). Collection can be triggeredby any combination of UV or mass spectrometry or can be based on time.Typical conditions for the separation process are as follows:Chromatography column was an Xbridge C-18 (19×100 mm); the gradient wasrun over a 7 minute period at a flow rate of 40 ml/min (gradient atstart: 10% MeOH and 90% water, gradient at finish: 100% MeOH and 0%water; as buffer either 0.1% formic acid, 0.1% ammonium hydroxide or0.1% TFA was added to the water). It will be appreciated by thoseskilled in the art that it may be necessary or desirable to modify theconditions for each specific compound, for example by changing thesolvent composition at the start or at the end, modifying the solventsor buffers, changing the run time, changing the flow rate and/or thechromatography column. Flash chromatography refers to silica gelchromatography and was carried out using an SP4 or an Isolera 4 MPLCsystem (manufactured by Biotage) and pre-packed silica gel cartridges(supplied by Biotage); or alternatively using conventional glass columnchromatography.

Analytical Methods

¹H Nuclear Magnetic Resonance (NMR) spectra were typically recordedusing an ECX400 spectrometer (manufactured by JEOL) in the statedsolvent at around rt unless otherwise stated. In all cases, NMR datawere consistent with the proposed structures. Characteristic chemicalshifts (δ) are given in parts-per-million using conventionalabbreviations for designation of major peaks: e.g. s, singlet; d,doublet; t, triplet; q, quartet; dd, doublet of doublets; br, broad.

Analytical LCMS was typically carried out using an Agilent HPLCinstrument with C-18 Xbridge column (3.5 μm, 4.6×30 mm, gradient atstart: 10% organic phase and 90% water, gradient at finish: organic and0% water; as buffer either 0.1% ammonium hydroxide or 0.1% TFA was addedto the water). The organic solvent was either acetonitrile or MeOH. Aflow rate of 3 mL/min was used with UV detection at 254 and 210 nm. Massspectra were recorded using a MM-ES+APCI mass spectrometer (G-1956A,manufactured by Agilent).

Compound Preparation

Where the preparation of starting materials is not described, these arecommercially available, known in the literature, or readily obtainableby those skilled in the art using standard procedures. Where it isindicated that compounds were prepared analogously to earlier examplesor intermediates, it will be appreciated by the skilled person that thereaction time, number of equivalents of reagents, solvent, concentrationand temperature can be modified for each specific reaction and that itmay be necessary or desirable to employ different work-up orpurification techniques.

Where reactions are carried out using microwave irradiation, themicrowave used is an Initiator 60 supplied by Biotage. The actual powersupplied varies during the course of the reaction in order to maintain aconstant temperature.

Some hydrogenations were carried out using an H-Cube® Continuous-flowHydrogenation Reactor manufactured by ThalesNano. The catalysts aresupplied by ThalesNano as “CatCarts” cartridges. The pressure, flowrate, temperature and cartridge are indicated in the experimentalsection. The equipment was used in accordance with the manufactureroperating procedure. The person skilled in the art will appreciate thatit may be necessary or desirable to run repeat cycles of the reactionmixture and in some instances, replace the cartridge between cycles toimprove the yield of the reaction.

Abbreviations

A list of some common abbreviations is shown below—where otherabbreviations are used which are not listed, these will be understood bythe person skilled in the art.

-   AcOH=Acetic acid-   BOC=tert-Butyloxycarbonyl-   CDI=1,1′-Carbonyldiimidazole-   Cs₂CO₃=Cesium carbonate-   DCM=Dichloromethane-   DIPEA=N,N-diisopropylethylamine-   DMF=N,N-Dimethytformamide-   DMSO=Dimethylsulfoxide-   EtOAc=Ethyl acetate-   EtOH=Ethanol-   HATU=N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)uranium    hexafluorophospate-   LCMS=Liquid Chromatography Mass Spectrometry-   LiCl=Lithium chloride-   MeOH=Methanol-   NaHCO₃=Sodium bicarbonate-   Na₂SO₄=Sodium sulfate-   NMP=N-Methylpyrrolidinone-   Pet ether=40160 petroleum ether-   Pd₂(dba)₃=tris(dibenzylideneacetone)dipalladium(0)-   rt=Room temperature-   SCX=Strong cation exchange-   TEA=Triethylamine-   TFA=Trifluoroacetic acid-   THF=Tetrahydrofuran-   Xantphos=4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene

The synthesis of selected compounds of the invention is described below.

1-(4-Bromo-3-nitrophenyl)piperazine (2)

To a solution of 1-(3-nitrophenyl)piperazine 1 (4.5 g, 21.7 mmol) inAcOH (100 mL) was added bromine (3.47 g, 21.7 mmol) at rt and stirredfor 16 h at 60° C. The reaction mixture was cooled to rt, theprecipitated solid was filtered, washed with water (2×50 mL) and driedto obtain compound 2 (4.5 g) as an orange crude solid; LCMS:(m/z)=286/288 [M+H]⁺. The crude solid was taken as such for next step.

tert-Butyl 4-(4-bromo-3-nitrophenyl)piperazine-1-carboxylate (3)

To a solution of 1-(4-bromo-3-nitrophenyl)piperazine 2 (4.5 g, 15.7mmol) in DCM (40 mL) was added TEA (4.7 g, 47.2 mmol) followed bydi-tert-butyl dicarbonate (5.1 g, 23.6 mmol) at rt and stirred for 16 h.The reaction mixture was concentrated in vacuo. The crude compound waspurified by column chromatography (silica gel, 100-200 mesh, eluted with10% EtOAc/pet ether) to obtain compound 3 (4.5 g, 75%) as a yellowsolid; ¹H NMR (300 MHz, CDCl₃) δ ppm 7.54 (d, J=9.3 Hz, 3H), 7.30 (d,J=3.0 Hz, 1H), 6.93 (dd, J=9.3, 3.0 Hz, 1H), 3.61-3.57 (m, 4H),3.22-3.19 (m, 4H), 1.48 (s, 9H); LCMS: (m/z)=286/288 [(M-BOC)+H]⁺.

tert-Butyl 4-(3-amino-4-bromophenyl)piperazine-1-carboxylate (4)

To a solution of tert-butyl4-(4-bromo-3-nitrophenyl)piperazine-1-carboxylate 3 (4.5 g, 11.6 mmol)in EtOH (40 mL) and water (8 mL) was added iron powder (1.95 g, 35.0mmol) at rt and heated to 50° C. Then ammonium chloride (3.7 g, 70.0mmol) was added at 50° C. and continued stirring for 2 h at 70° C. Thereaction mixture was cooled to rt and water (20 mL) was added. Themixture was filtered through a pad of celite and the filtrate wasextracted with EtOAc (2×40 mL). The combined organic layers were dried(Na₂SO₄) and concentrated in vacuo. The crude compound was purified bytriturating with n-pentane to obtain compound 4 (3.5 g, 85%) as an offwhite solid; ¹H NMR (300 MHz, CDCl₃) δ ppm 7.25 (d, J=8.7 Hz, 1H), 6.31(d, J=3.0 Hz, 1H), 6.24 (dd, J=8.7, 3.0 Hz, 1H), 4.10 (br s, 2H),3.56-3.53 (m, 4H), 3.08-3.05 (m, 4H), 1.48 (s, 9H); LCMS: (m/z)=356/358[M+H]⁺.

tert-Butyl4-(3-(2-aminooxazole-4-carboxamido)-4-bromophenyl)piperazine-1-carboxylate(5)

To a solution of tert-butyl4-(3-amino-4-bromophenyl)piperazine-1-carboxylate 4 (3.5 g, 9.83 mmol)in DMF (20 mL) was added 2-aminooxazole-4-carboxylic acid (5.0 g, 39.3mmol), HATU (14.9 g, 39.3 mmol) followed by DIPEA (3.8 g, 29.5 mmol) atrt and stirred for 16 h. Ice cold water (50 mL) was added and stirringcontinued for 10 min. The precipitate was filtered, dried and purifiedby column chromatography (silica gel, 100-200 mesh, eluted with 50%EtOAc/pet ether) to obtain compound 6 (2.5 g, 55%) as light yellowsolid; LCMS: (m/z)=466/468 [M+H]⁺.

2-Amino-N-(2-bromo-5-(piperazin-1-yl)phenyl)oxazole-4-carboxamide (6)

To a solution of tert-butyl4-(3-(2-aminooxazole-4-carboxamido)-4-bromophenyl)piperazine-1-carboxylate5 (0.5 g, 1.07 mmol) in DCM (5 mL) was added TFA (5 mL) at rt and thereaction stirred for 2 h. The reaction mixture was concentrated invacuo, the obtained residue was dissolved in water (10 mL) and basifiedwith saturated NaHCO₃ solution. The precipitate was filtered, dried andpurified by triturating with saturated NaHCO₃ solution to obtaincompound 6 (0.32 g, 82%) as an off white solid; ¹H NMR (300 MHz,DMSO-d₆) δ ppm 9.13 (s, 1H), 8.76 (br, s, 1H), 8.06 (s, 1H), 8.01 (d,J=3.0 Hz, 1H), 7.52 (d, J=8.7 Hz, 1H), 7.14 (s, 2H), 6.78 (dd, J=8.7,3.0 Hz, 1H), 3.34 (br s, 4H), 3.23 (br s, 4H); LCMS: (m/z)=366/368[M+H]⁺.

The following intermediate was prepared using analogous procedures:

2-Amino-N-[2-chloro-6-(piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(7)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.13 (s, 1H), 8.06 (s, 1H), 7.98 (d,J=2.9 Hz, 1H), 7.31 (d, J=8.8 Hz, 1H), 7.15 (s, 2H), 6.73 (br dd, J=8.8,2.4 Hz, 1H), 3.09-3.00 (m, 4H), 2.82 (br s, 4H); LCMS (m/z): 322/324[M+H]⁺.

tert-Butyl 4-[4-(methylsulfanyl)-3-nitrophenyl]piperazine-1-carboxylate(9)

tert-Butyl piperazine-1-carboxylate (0.56 g, 3.00 mmol),4-bromo-1-(methylsulfanyl)-2-nitrobenzene 8 (750 mg, 3.00 mmol),palladium (II) acetate (54 mg, 0.24 mmol), Xantphos (210 mg, 0.36 mmol)and Cs₂CO₃ (2 g, 6.10 mmol) were combined in DMF (10 mL) and stirred at110° C. for 3 h. The reaction mixture was filtered through a silicaplug, diluted with EtOAc (100 mL), washed with water (3×100 mL) and thensaturated LiCl (aq) solution (3×15 mL). The organics were dried,concentrated in vacuo and purified by Biotage Isolera, eluting with0-14% EtOAc/pet ether to obtain compound 9 (0.78 g, 73%) as an orangesolid; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.63 (d, J=2.3 Hz, 1H), 7.45-7.39(m, 2H), 3.50-3.42 (m, 4H), 3.22-3.16 (m, 4H), 2.47 (s, 3H), 1.41 (s,9H); LCMS (m/z): 354 [M+H]⁺.

tert-Butyl 4-[3-amino-4-(methylsulfanyl)phenyl]piperazine-1-carboxylate(10)

tert-Butyl 4-[4-(methylsulfanyl)-3-nitrophenyl]piperazine-1-carboxylate9 (550 mg, 1.56 mmol) was dissolved in EtOAc (70 mL) and EtOH (40 mL)and hydrogenated using an H-cube with 10% Pd/C at 30° C. This processwas repeated, the eluent concentrated in vacuo, and the residue purifiedby Isolute-SCX cartridge to obtain compound 10 (510 mg, 100%) as a brownsolid; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.09 (d, J=8.7 Hz, 1H), 6.28 (d,J=2.5 Hz, 1H), 6.18 (dd, J=8.7, 2.5 Hz, 1H), 5.15 (br s, 2H), 3.46-3.38(m, 4H), 3.13-2.99 (m, 4H), 2.17 (s, 3H), 1.41 (s, 9H); LCMS (m/z): 324[M+H]⁺.

tert-Butyl4-[3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-(methylsulfanyl)phenyl]piperazine-1-carboxylate(11)

2-Amino-1,3-oxazole-4-carboxylic acid (0.27 g, 2.11 mmol), HATU (0.82 g,2.11 mmol) and DIPEA (2.20 mL, 12.6 mmol) were added to tert-butyl4-[3-amino-4-(methylsulfanyl)phenyl]piperazine-1-carboxylate 10 (680 mg,2.11 mmol) in DMF (20 mL) and the reaction mixture was stirred at rt for18 h. The mixture was partitioned between EtOAc (15 mL) and water (50mL). The organic layer was washed with water (2×50 mL), saturated LiCl(aq) (3×15 mL), dried, concentrated in vacuo and purified by columnchromatography (eluting with 10-100% EtOAc/pet ether) to obtain compound11 (0.40 g, 44%) as a brown solid; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.66(s, 1H) 8.09 (d, J=2.7 Hz, 1H) 8.04 (s, 1H) 7.42 (d, J=8.7 Hz, 1H) 7.15(s, 2H) 6.73 (dd, J=8.7, 2.7 Hz, 1H) 3.41-3.50 (m, 4H) 3.09-3.18 (m, 4H)2.30 (s, 3H) 1.41 (s, 9H); LCMS (m/z): 434 [M+H]⁺.

2-Amino-N-[2-(methylsulfanyl)-5-(piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(12)

To a solution oftert-butyl4-[3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-(methylsulfanyl)phenyl]piperazine-1-carboxylate11 (0.40 g, 0.92 mmol) in DCM (4 mL) was added TFA (4 mL) at rt and thereaction stirred for 2 h. The reaction mixture was concentrated invacuo, and then purified using an Isolute-SCX cartridge to obtaincompound 12 (270 mg, 88%) as an orange solid; ¹H NMR (400 MHz, DMSO-d₆)δ ppm 9.67 (s, 1H), 8.08 (d, J=2.7 Hz, 1H), 8.03 (s, 1H), 7.39 (d, J=8.7Hz, 1H), 7.15 (s, 2H), 6.69 (dd, J=8.7, 2.7 Hz, 1H), 3.34 (s, 1H),3.10-3.02 (m, 4H), 2.87-2.76 (m, 4H), 2.29 (s, 3H); LCMS (m/z): 334[M+H]⁺.

2-Amino-N-(2-bromo-5-{4-[(3-chloropropyl)sulfonyl]piperazin-1-yl}phenyl)-1,3-oxazole-4-carboxamide(13)

To a stirred solution of2-amino-N-(2-bromo-5-(piperazin-1-yl)phenyl)oxazole-4-carboxamide 6 (12g, 32.9 mmol) in DMF (120 mL) was added 3-chloropropane-1-sulfonylchloride (7.97 mL, 65.8 mmol), TEA (13.7 mL, 98.0 mmol) at 0° C. andstirred at rt for 6 h. The reaction mixture was poured into cold water(200 mL), filtered the solid and dried to obtain compound 13 (12 g, 72%)as a pale yellow solid; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H),8.07 (s, 1H), 7.99 (d, J=2.8 Hz, 1H), 7.91 (s, 1H), 7.49 (d, J=9.2 Hz,1H), 7.15 (s, 2H), 6.76-6.63 (m, 1H), 3.75 (t, J=6.6 Hz, 2H), 3.34-3.31(m, 2H), 3.25-3.17 (m, 6H), 3.18-3.11 (m, 2H), 2.18-2.11 (m, 2H); LCMS:(m/z) 506/508/510 [M+H]⁺.

The following intermediates were prepared using an analogous procedure:

2-Amino-N-(2-chloro-5-{4-[(3-chloropropyl)sulfonyl]piperazin-1-yl}phenyl)-1,3-oxazole-4-carboxamide(14)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.06 (s, 1H), 8.01 (d,J=3.2 Hz, 1H), 7.36 (d, J=9.2 Hz, 1H), 7.15 (s, 2H), 6.79 (dd, J=9.2,3.2 Hz, 1H), 3.74 (s, 2H), 3.34-3.30 (m, 4H), 3.26-3.19 (m, 6H),2.17-2.10 (m, 2H); LCMS: (m/z)=462/464/466 [M+H]⁺.

2-Amino-N-(2-bromo-5-{4-[(3-chloro-2-methylpropyl)sulfonyl]piperazin-1-yl}phenyl)-1,3-oxazole-4-carboxamide(15)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=3.2 Hz, 1H), 7.49 (d, J=8.9 Hz, 1H), 7.16 (s, 2H), 6.75 (dd, J=8.9,3.2 Hz, 1H), 3.73 (d, J=5.0 Hz, 2H), 3.33-3.27 (m, 4H), 3.27-3.19 (m,5H), 3.02 (dd, J=14.2, 7.3 Hz, 1H), 2.45-2.35 (m, 1H), 1.15 (d, J=6.4Hz, 3H); LCMS (m/z): 520/522 [M+H]⁺.

2-Amino-N-{2-bromo-6-[4-(ethenylsulfonyl)piperazin-1-yl]phenyl}-1,3-oxazole-4-carboxamide(16)

2-Amino-N-[2-bromo-5-(piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide 6(1 g, 2.73 mmol) and TEA (1.14 mL, 8.19 mmol) were combined in DMF (10mL) at 0° C. The reaction was stirred at 0° C. for 5 minutes, then2-chloroethanesulfonyl chloride (0.28 mL, 2.73 mmol) was added and thereaction mixture was allowed to warm to rt over 5 h. The mixture wasthen partitioned between EtOAc and saturated LiCl (aq), the organiclayer recovered using a phase separation cartridge and concentrated invacuo. The crude product was purified by column chromatography (elutingwith 0-60% EtOAc/pet ether gradient) to give the desired product 16(0.22 g, 18%); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.07 (s,1H), 7.97 (d, J=3.2 Hz, 1H), 7.49 (d, J=8.7 Hz, 1H), 7.14 (br s, 2H),6.86 (dd, J=16.5, 10.1 Hz, 1H), 6.74 (dd, J=8.9, 3.0 Hz, 1H), 6.22 (d,J=10.1 Hz, 1H), 6.15 (d, J=16.5 Hz, 1H), 3.28-3.22 (m, 4H), 3.20-3.14(m, 4H); LCMS: (m/z)=456/458 [M+H]⁺.

The following intermediate was prepared using an analogous procedure:

2-Amino-N-{2-chloro-5-[4-(ethenylsulfonyl)piperazin-1-yl]phenyl}-1,3-oxazole-4-carboxamide(17)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.08-8.06 (m, 1H), 7.99(d, J=2.7 Hz, 1H), 7.35 (d, J=8.9 Hz, 1H), 7.15 (br s, 2H), 6.87 (dd,J=16.5, 10.1 Hz, 1H), 6.79 (dd, J=8.9, 3.0 Hz, 1H), 6.24-6.13 (m, 2H),3.25 (dd, J=6.2, 3.0 Hz, 4H), 3.21-3.13 (m, 4H); LCMS (m/z): 412/414[M+H]⁺.

Azetidin-3-ylmethyl 4-(3-(2-aminooxazole-4-carboxamido)-4-chlorophenyl)piperazine-1-carboxylate (19)

To a stirred solution of tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate (815 mg, 4.35 mmol) in THF (10 mL) was added CDI(1.14 g, 4.35 mmol) at 0° C., and stirred for 30 minutes.2-Amino-N-[2-chloro-5-(piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide7 (700 mg, 2.18 mmol) and TEA (0.6 mL, 4.35 mmol) were added and thereaction mixture was stirred at rt for 16 h. The reaction mixture wasdiluted with water (20 mL) and extracted with EtOAc (2×25 mL). Thecombined organics were washed with brine (10 mL), dried (Na₂SO₄) andconcentrated in vacuo. Purification by column chromatography (elutingwith 50% EtOAc/pet ether) gave compound 18 (500 mg, 44%) as a brownsolid; LCMS (m/z): 535/537 [M+H]⁺. This was re-dissolved in DCM (10 mL),treated with TFA (3 mL) at 0° C., and stirred at rt for 16 h. Thereaction mixture was concentrated in vacuo. The obtained residue wasbasified with saturated NaHCO₃ (aq) solution (10 mL) at 0° C. andextracted with EtOAc (2×50 mL). The combined organics were washed withbrine (10 mL), dried (Na₂SO₄) and concentrated in vacuo. DCM (3×5 mL)was added to the residue and the resultant solid was filtered, washedwith diethyl ether and dried to obtain 19 (250 mg, 62%) as a brownsolid; ¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.06 (s, 1H), 7.99(m, 1H), 7.34 (d, J=8.7 Hz, 1H), 7.14 (br s, 2H), 6.78 (d, J=6.3 Hz,1H), 4.11 (d, J=5.7 Hz, 1H), 3.63 (t, J=8.1 Hz, 1H), 3.51 (m, 4H), 3.32(m, 4H), 3.13 (m, 4H); LCMS (m/z): 435/437 [M+H]⁺.

(1-Methylazetidin-3-yl) methyl4-(3-(2-aminooxazole-4-carboxamido)-4-chlorophenyl)piperazine-1-carboxylate (20)

To a stirred solution of azetidin-3-ylmethyl4-(3-(2-aminooxazole-4-carboxamido)-4-chlorophenyl)piperazine-1-carboxylate 19 (200 mg, 0.46 mmol) in MeOH (5 mL) was added30% formaldehyde (aq) (20 mg, 0.66 mmol) and zinc chloride (188 mg, 1.38mmol) at 0° C., and stirred at rt for 2 h. Sodium cyanoborohydride (86mg, 1.38 mmol) was then added at 0° C., and stirred at rt for 16 h. Thereaction mixture was basified with saturated NaHCO₃ (aq) solution (5 mL)and concentrated in vacuo. The obtained residue was dissolved in MeOH(25 mL) and filtered through Celite and the filtrate was concentrated invacuo. Purification by preparative HPLC gave 20 (25 mg, 12%) as an offwhite solid; ¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.06 (s, 1H),7.99 (d, J=2.8 Hz, 1H), 7.34 (d, J=8.8 Hz, 1H), 7.13 (br s, 2H),6.78-6.75 (m, 1H), 4.12 (d, J=6.8 Hz, 2H), 3.52 (m, 4H), 3.22 (t, J=7.2Hz, 2H), 3.13 (m, 4H), 2.87 (t, J=6.4 Hz, 1H), 2.62-2.59 (m, 1H), 2.17(s, 3H); LCMS (m/z): 449/451 [M+H]⁺.

The following examples were prepared from intermediates 6, 7 or 12 usinganalogous procedures:

2-(Cyclopropylamino)ethyl4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-bromophenyl)piperazine-1-carboxylate(21)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.06 (s, 1H), 7.98 (d,J=2.9 Hz, 1H), 7.48 (d, J=8.8 Hz, 1H), 7.13 (br s, 2H), 6.73 (dd, J=2.9,9.3 Hz, 1H), 4.08 (br t, J=5.6 Hz, 2H), 3.52 (br s, 4H), 3.15 (br d,J=4.4 Hz, 4H), 2.85 (br t, J=5.6 Hz, 2H), 2.17 (br s, 1H), 0.40 (br d,J=4.4 Hz, 2H), 0.26 (br s, 2H); LCMS (m/z)=493/495 [M+H]⁺.

Azetidin-3-yl4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-chlorophenyl)piperazine-1-carboxylate(22)

¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.07 (s, 1H), 8.00 (d,J=2.6 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.15 (s, 2H), 6.77 (dd, J=2.8,9.0 Hz, 1H), 5.07 (br s, 1H), 3.65-3.42 (m, 9H), 3.15 (br d, J=4.8 Hz,4H); LCMS: (m/z)=421/423 [M+H]⁺.

2-(Dimethylamino)ethyl4-(3-(2-aminooxazole-4-carboxamido)-4-bromophenyl)piperazine-1-carboxylate(23)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.09 (s, 1H), 8.06 (s, 1H), 7.97 (d,J=2.4 Hz, 1H), 7.46 (d, J=8.8 Hz, 1H), 7.13 (s, 2H), 6.71 (dd, J=8.8,2.4 Hz, 1H), 4.10 (t, J=5.2 Hz, 2H), 3.50 (br s, 4H), 3.13 (br s, 4H),2.47 (m, 2H), 2.17 (s, 6H); LCMS: (m/z)=481/483 [M+H]⁺.

3-(Dimethylamino)propyl4-(3-(2-aminooxazole-4-carboxamido)-4-bromophenyl)piperazine-1-carboxylate(24)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.09 (s, 1H), 8.06 (s, 1H), 7.98 (d,J=3.2 Hz, 1H), 7.47 (d, J=8.8 Hz, 1H), 7.13 (s, 2H), 6.72 (dd, J=2.8,9.2 Hz, 1H), 4.04 (t, J=6.4 Hz, 2H), 3.51 (br s, 4H), 3.14 (br t, J=4.8Hz, 4H), 2.26 (t, J=6.8 Hz, 2H), 2.12 (s, 6H), 1.74-1.67 (m, 2H); LCMS:(m/z)=495/497 [M+H]⁺.

2-(Methylamino)ethyl4-(3-(2-aminooxazole-4-carboxamido)-4-bromophenyl)piperazine-1-carboxylate(25)

¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.06 (s, 1H), 7.98 (d,J=2.6 Hz, 1H), 7.47 (d, J=8.8 Hz, 1H), 7.13 (s, 2H), 6.73 (dd, J=9.0,2.8 Hz, 1H), 4.05 (t, J=5.5 Hz, 2H), 3.52 (br s, 4H), 3.15 (br d, J=4.8Hz, 4H), 2.69 (t, J=5.7 Hz, 2H), 2.29 (s, 3H); LCMS (m/z)=467/469[M+H]⁺.

3-(Dimethylamino)propyl-4-[3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-(methylsulfanyl)phenyl]piperazine-1-carboxylate(26)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.66 (s, 1H), 8.09 (d, J=2.7 Hz, 1H),8.04 (s, 1H), 7.43 (d, J=8.7 Hz, 1H), 7.15 (br s, 2H), 6.73 (dd, J=8.7,2.7 Hz, 1H), 4.04 (t, J=6.6 Hz, 2H), 3.55-3.44 (m, 4H), 3.19-3.12 (m,4H), 2.43-2.36 (m, 2H), 2.30 (s, 3H), 2.22 (s, 6H), 1.79-1.71 (m, 2H);LCMS (m/z): 463 [M+H]⁺.

2-(Dimethylamino)ethyl4-[3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-(methylsulfanyl)phenyl]piperazine-1-carboxylate(27)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.66 (s, 1H), 8.09 (d, J=2.7 Hz, 1H),8.04 (s, 1H), 7.43 (d, J=8.7 Hz, 1H), 7.15 (s, 2H), 6.73 (dd, J=8.7, 2.7Hz, 1H), 4.04 (t, J=6.6 Hz, 2H), 3.55-3.45 (m, 4H), 3.19-3.12 (m, 4H),2.40 (t, J=7.1 Hz, 2H), 2.30 (s, 3H) 2.22 (s, 6H); LCMS (m/z): 449[M+H]⁺.

1-Methylazetidin-3-yl4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-bromophenyl)piperazine-1-carboxylate(28)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.06 (s, 1H), 7.97 (d,J=2.9 Hz, 1H), 7.48 (d, J=8.8 Hz, 1H), 7.13 (s, 2H), 6.72 (dd, J=8.8,2.9 Hz, 1H), 4.83 (quintet, J=5.7 Hz, 1H), 3.58-3.46 (m, 6H), 3.18-3.11(m, 4H), 2.96-2.90 (m, 2H), 2.24 (s, 3H); LCMS (m/z)=479/481 [M+H]⁺.

3-(Dimethylamino)propyl4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-chlorophenyl)piperazine-1-carboxylate(29)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.07 (s, 1H), 8.00 (d,J=2.9 Hz, 1H), 7.35 (d, J=9.3 Hz, 1H), 7.15 (s, 2H), 6.78 (dd, J=9.0,2.7 Hz, 1H), 4.05 (t, J=6.6 Hz, 2H), 3.51 (br s, 4H), 3.18-3.10 (m, 4H),2.34 (br d, J=6.8 Hz, 2H), 2.18 (s, 6H), 1.73 (quintet, J=6.8 Hz, 2H);LCMS: (m/z)=451/453 [M+H]⁺.

1-Methylpyrrolidin-3-yl4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-chlorophenyl)piperazine-1-carboxylate(30)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=2.7 Hz, 1H), 7.35 (d, J=9.3 Hz, 1H), 7.14 (br s, 2H), 6.77 (dd, J=8.7,2.7 Hz, 1H), 5.03-4.99 (m, 1H), 3.51-3.48 (m, 4H), 3.14-3.11 (m, 4H),2.68-2.48 (m, 3H), 2.28-2.15 (m, 5H), 1.76-1.66 (m, 1H); LCMS (m/z):449/451 [M+H]⁺.

2-(Methylamino)ethyl4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}4-chlorophenyl)piperazine-1-carboxylate(31)

¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.06 (s, 1H), 8.00 (d,J=2.6 Hz, 1H), 7.35 (d, J=9.2 Hz, 1H), 7.14 (s, 2H), 6.77 (dd, J=8.8,2.9 Hz, 1H), 4.05 (t, J=5.5 Hz, 2H), 3.52 (br s, 4H), 3.17-3.10 (m, 4H),2.68 (t, J=5.7 Hz, 2H), 2.29 (s, 3H); LCMS (m/z)=423/425 [M+H]⁺.

Pyrrolidin-3-yl4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-chlorophenyl)piperazine-1-carboxylate(32)

¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.07 (s, 1H), 8.00 (d,J=3.0 Hz, 1H), 7.35 (d, J=9.3 Hz, 1H), 7.15 (s, 2H), 8.77 (dd, J=6.0,3.0 Hz, 1H), 5.07 (t, J=5.4 Hz, 1H), 3.49 (s, 4H), 3.12 (s, 4H)2.94-2.80 (m, 2H), 2.75-2.49 (m, 2H), 1.92-1.85 (m, 1H), 1.70-1.66 (m,1H); LCMS: (m/z)=435/437 [M+H]⁺.

2-(Dimethylamino)ethyl4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-chlorophenyl)piperazine-1-carboxylate(33)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=2.9 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.15 (s, 2H), 6.77 (dd, J=8.8,2.9 Hz, 1H), 4.10 (t, J=5.9 Hz, 2H), 3.51 (br s, 4H), 3.17-3.09 (m, 4H),2.47 (br s, 2H), 2.17 (s, 6H); LCMS: (m/z)=437/439 [M+H]⁺.

1-Methylazetidin-3-yl4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-chlorophenyl)piperazine-1-carboxylate(34)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=2.4 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.15 (s, 2H), 6.77 (dd, J=8.8,2.9 Hz, 1H), 4.85-4.82 (m, 1H), 3.56-3.51 (m, 6H), 3.15-3.12 (m, 4H),2.50-2.49 (m, 2H), 2.24 (s, 3H); LCMS: (m/z)=435/437 [M+H]⁺.

2-(Cyclopropylamino)ethyl4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-chlorophenyl)piperazine-1-carboxylate(35)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.06 (s, 1H), 8.00 (d,J=2.4 Hz, 1H), 7.35 (d, J=9.3 Hz, 1H), 7.14 (s, 2H), 6.77 (dd, J=9.3,2.9 Hz, 1H), 4.06 (t, J=5.9 Hz, 2H), 3.52 (br s, 4H), 3.17-3.09 (m, 4H),2.79 (br t, J=5.6 Hz, 2H), 2.33 (br d, J=1.5 Hz, 1H), 2.10 (tt, J=6.7,3.5 Hz, 1H), 0.38-0.31 (m, 2H), 0.22-0.15 (m, 2H); LCMS (m/z): 449/451[M+H]⁺.

Benzyl 4-((1-(tert-butoxycarbonyl) azetidin-3-yl)methyl)-3-oxopiperazine-1-carboxylate (37)

To a solution of benzyl 3-oxopiperazine-1-carboxylate (10 g, 42.7 mmol)in NMP (80 mL) was added 60% sodium hydride (4.25 g, 106 mmol) at 0° C.and stirred for 1 h. Then slowly added a solution of tert-butyl3-((methylsulfonyloxy)methyl)azetidine-1-carboxylate 36 (16.9 g, 63.8mmol) in NMP (20 mL) over a period of 15 minutes and stirred 30 minutesat 0° C., then allowed to warm to rt and stirred for 16 h. The reactionmixture was poured into ice water (500 mL) and extracted with EtOAc(2×500 mL). The combined organic layer was washed with brine solution(100 mL), dried (Na₂SO₄) and concentrated in vacuo. Purification bycolumn chromatography (eluting with 60% EtOAc/pet ether) gave 37 (2.5 g)as a white solid; ¹H NMR (300 MHz, CDCl₃) δ ppm 7.35 (s, 5H), 5.15 (s,2H), 4.15 (s, 2H), 3.99 (t, J=8.4 Hz, 2H), 3.72-3.64 (m, 6H), 3.35 (m,2H), 2.90-2.82 (m, 1H), 1.42 (s, 9H); LCMS (m/z): 404 [M+H]⁺.

tert-Butyl 3-((2-oxopiperazin-1-yl)methyl)azetidine-1-carboxylate (38)

To a solution of benzyl 4-((1-(tert-butoxycarbonyl) azetidin-3-yl)methyl)-3-oxopiperazine-1-carboxylate 37 (2.5 g, 6.20 mmol) in MeOH (50mL) was added 10% Pd/C (500 mg) under N₂ atmosphere. The reactionmixture was hydrogenated under a balloon of hydrogen and stirred at rtfor 4 h. The reaction mixture was filtered through a Celite pad, washedwith MeOH (25 mL) and the filtrate was concentrated in vacuo to obtaincompound 38 (1.5 g, 89%) as a black gummy solid; ¹H NMR (400 MHz, CDCl₃)δ ppm 4.00 (t, J=8.4 Hz, 2H), 3.71-3.67 (m, 3H), 3.52 (m, 2H), 3.31 (t,J=5.2 Hz, 2H), 3.07 (t, J=5.2 Hz, 2H), 2.86-2.82 (m, 1H), 1.43 (s, 9H);LCMS (m/z): 270 [M+H]⁺.

tert-Butyl 3-((4-(4-bromo-3-(tert-butoxycarbonylamino)phenyl)-2-oxopiperazin-1-yl) methyl) azetidine-1-carboxylate (39)

In a sealed tube, to a degassed solution of tert-butyl2-bromo-5-iodophenylcarbamate (1.0 g, 2.51 mmol) in 1,4-dioxane (15 mL),Cs₂CO₃ (1.63 g, 5.01 mmol), tort-butyl3-((2-oxopiperazin-1-yl)methyl)azetidine-1-carboxylate 38 (811 mg, 3.01mmol) and Xantphos (145 mg, 0.25 mmol) was added Pd₂(dba)₃ (114 mg, 0.12mmol) at rt and again degassed for 10 min (argon). The reaction mixturewas heated at 100° C. for 16 h. The reaction mixture was cooled to rt,filtered through Celite, washed with EtOAc (50 mL), and the filtrateconcentrated in vacuo. Purification by column chromatography (elutingwith 60% EtOAc/pet ether gave 39 (800 mg, 59%) as a gummy liquid; ¹H NMR(400 MHz, CDCl₃) δ ppm 7.83 (d, J=2.4 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H),6.98 (s, 1H), 6.40-6.37 (m, 1H), 4.14 (m, 1H), 4.01 (t, J=8.4 Hz, 2H),3.99 (s, 2H), 3.72-3.68 (m, 3H), 3.47 (s, 4H), 2.88-2.85 (m, 1H), 1.54(s, 9H), 1.43 (s, 9H); LCMS (m/z): 539/541 [M+H]⁺.

tert-Butyl3-((4-(3-amino-4-bromophenyl)-2-oxopiperazin-1-yl)methyl)azetidine-1-carboxylate(41)

To a solution of tert-butyl 3-((4-(4-bromo-3-(tert-butoxycarbonylamino)phenyl)-2-oxopiperazin-1-yl) methyl) azetidine-1-carboxylate 39 (800 mg,1.48 mmol) in DCM (10 mL) was added TFA (5 mL) at 0° C., and stirred atrt for 16 h. The reaction mixture was concentrated in vacuo to obtaincompound 40 as a TFA salt (800 mg, crude) as a gummy liquid.

To a solution of 40 (800 mg, 1.41 mmol) in MeOH (10 mL) was added TEA(0.8 mL 5.65 mmol) and di-tert-butyl dicarbonate (462 mg, 2.12 mmol) at0° C., and stirred at rt for 16 h. The reaction mixture was concentratedin vacuo, and then purified by column chromatography (eluting with 70%EtOAc/pet ether) to obtain compound 41 (200 mg, 30% over two steps) as apale yellow solid; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.14 (d, J=8.8 Hz,1H), 6.33 (d, J=4.0 Hz, 1H), 6.14-6.11 (m, 1H), 5.09 (s, 2H), 3.87 (m,2H), 3.67 (s, 2H), 3.57 (m, 4H), 3.38-3.36 (m, 4H), 2.82-2.72 (m, 1H),1.37 (s, 9H); LCMS (m/z): 439 [M+H]⁺.

2-Amino-N-(2-bromo-6-{4-[(1-methylazetidin-3-yl)methyl]-3-oxopiperazin-1-yl}phenyl)-1,3-oxazole-4-carboxamide(44)

tert-Butyl 3-((4-(3-amino-4-bromophenyl)-2-oxopiperazin-1-yl) methyl)azetidine-1-carboxylate 41 (200 mg, 0.45 mmol),2-aminooxazole-4-carboxylic acid (204 mg, 1.59 mmol), HATU (607 mg, 1.59mmol) and DIPEA (0.3 mL, 1.59 mmol) were combined in DMF (2 mL) at 0° C.and stirred at rt for 16 h. The reaction mixture was diluted with water(10 mL), the solid filtered, washed with diethyl ether (5 mL) and driedto obtain compound 42 (200 mg, crude) as a brown solid. This wasre-dissolved in DCM (3 mL), treated with TFA (1 mL) at 0° C., andstirred at rt for 16 h. The reaction mixture was concentrated in vacuo,the obtained residue was co-distilled with DCM (2×10 mL) and dried toobtain compound 43 (200 mg, crude) as a gummy liquid.

A solution of 43 (200 mg, 0.44 mmol) in MeOH (5 mL) was treated with 30%formaldehyde (aq) (0.08 mL, 0.86 mmol) and sodium acetate (109 mg, 1.32mmol) at 0° C., and stirred at rt for 1 h. Sodium cyanoborohydride (83mg, 1.33 mmol) was then added at 0° C., and stirred at rt for 16 h. Thereaction mixture was concentrated in vacuo, dissolved in 10% MeOH/DCM(25 mL), filtered and dried. The obtained residue was purified bypreparative HPLC to obtain 44 (22 mg, 10% over 3 steps) as a pale yellowsolid; ¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.06 (s, 1H), 7.93(d, J=3.0 Hz, 1H), 7.47 (d, J=8.7 Hz, 1H), 7.14 (s, 2H), 6.69 (dd,J=8.7, 3.0 Hz, 1H), 3.77 (s, 2H), 3.54 (d, J=7.2 Hz, 2H), 3.43 (br s,4H), 3.22 (t, J=7.2 Hz, 2H), 2.79 (t, J=6.3 Hz, 2H), 2.59-2.54 (m, 1H),2.15 (s, 3H); LCMS (m/z): 463/465 [M+H]⁺.

The following examples were prepared using analogous procedures:

2-Amino-N-(2-bromo-6-{4-[2-(dimethylamino)ethyl]-3-oxopiperazin-1-yl}phenyl)-1,3-oxazole-4-carboxamide(45)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.06 (s, 1H), 7.93 (d,J=2.8 Hz, 1H), 7.47 (d, J=5.2 Hz, 1H), 7.13 (s, 2H), 6.71-6.68 (m, 1H),3.76 (s, 2H), 3.48-3.44 (m, 6H), 2.39 (t, J=6.4 Hz, 2H), 2.16 (s, 6H);LCMS (m/z): 451/453 [M+H]⁺.

2-Amino-N-(2-chloro-5-{4-[3-(dimethylamino)propyl]-3-oxopiperazin-1-yl}phenyl)-1,3-oxazole-4-carboxamide(46)

¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 7.96 (d,J=3.0 Hz, 1H), 7.35 (d, J=9.3 Hz, 1H), 7.17 (s, 2H), 6.77 (m, 1H), 3.78(s, 2H), 3.47 (s, 4H), 3.38 (m, 2H), 2.23 (t, J=6.9 Hz, 2H), 2.15 (s,6H), 1.69-1.62 (m, 2H); LCMS (m/z): 421/423 [M+H]⁺.

tert-Butyl2-(4-(4-chloro-3-nitrophenyl)piperazin-1-ylsulfonyl)ethylcarbamate (50)

To a solution of 1-(4-chloro-3-nitrophenyl)piperazine (3.0 g, 12.4 mmol)in DCM (20 mL) was added TEA (2.5 g, 24.9 mmol) followed by benzyl2-(chlorosulfonyl)ethylcarbamate 47 (15 g, crude) at 0° C., and stirredat rt for 16 h. The reaction mixture was diluted with water (100 mL) andextracted with DCM (2×100 mL). The organics were washed with water (2×50mL), brine (25 mL), dried (Na₂SO₄) and concentrated in vacuo.Purification by column chromatography (eluting with 30% EtOAc/pet ether)gave compound 48 (2 g) as an impure pale yellow liquid. This was takento the next step without any purification. A solution of 48 (2 g, 4.14mmol) in 30% hydrogen bromide in AcOH (6 mL) was stirred at rt for 6 h.The reaction mixture was concentrated in vacuo. The obtained residue wasbasified with saturated NaHCO₃ (aq) (20 mL) and extracted with EtOAc(2×25 mL). The organics were washed with brine (10 mL), dried (Na₂SO₄)and concentrated in vacuo to obtain the crude product 49 (1 g) as ayellow liquid.

A solution of 49 (1 g, 2.87 mmol) in DCM (20 mL) was treated with TEA(0.8 mL, 5.76 mmol) and di-tert-butyl dicarbonate (689 mg, 3.16 mmol) at0° C., and stirred at rt for 16 h. The reaction mixture was diluted withwater (25 mL) and extracted with DCM (2×50 mL). The organics were washedwith brine (25 mL), dried (Na₂SO₄) and concentrated in vacuo to obtaincompound 50 (750 mg, crude) as a yellow liquid; ¹H NMR (300 MHz, CDCl₃)δ ppm 7.40 (d, J=8.8 Hz, 1H), 7.36 (d, J=2.8 Hz, 1H), 7.04-7.01 (m, 1H),3.61 (t, J=5.6 Hz, 2H), 3.45 (t, J=5.2 Hz, 4H), 3.32 (t, J=5.2 Hz, 4H);3.16 (t, J=6.0 Hz, 2H), 1.44 (s, 9H); LCMS (m/z): 393[(M−56)+H]⁺. Thecrude compound was taken as such for the next step without anypurification.

tert-Butyl2-(4-(3-amino-4-chlorophenyl)piperazin-1-ylsulfonyl)ethylcarbamate (51)

tert-Butyl2-(4-(4-chloro-3-nitrophenyl)piperazin-1-ylsulfonyl)ethylcarbamate 50(750 mg, 1.67 mmol), NH₄Cl (552 mg, 10.0 mmol) and iron (266 mg, 5.01mmol) were combined in EtOH (15 mL)/H₂O (10 mL). The reaction mixturewas heated at 80° C. for 6 h, and then allowed to cool, filtered throughCelite, washed with MeOH (25 mL), and the filtrate was concentrated invacuo. The obtained residue was diluted with water (20 mL) and stirredfor 10 minutes. Resultant solid was filtered, washed with water anddried in vacuo to obtain compound 51 (300 mg, 43%) as a brown solid; ¹HNMR (300 MHz, DMSO-d₆) δ ppm 7.01-6.95 (m, 2H), 6.38 (d, J=2.4 Hz, 1H),6.22-6.19 (m, 1H), 5.14 (s, 2H), 3.31-3.25 (m, 6H), 3.20-3.10 (m, 6H),1.37 (s, 9H); LCMS (m/z): 419/421 [M+H]⁺.

tert-Butyl2-(4-(3-(2-aminooxazole-4-carboxamido)-4-chlorophenyl)piperazin-1-ylsulfonyl)ethylcarbamate (52)

To a solution of tert-butyl 2-(4-(3-amino-4-chlorophenyl)piperazin-1-ylsulfonyl)ethylcarbamate 51 (300 mg, 0.717 mmol) in DMF (3mL) was added 2-aminooxazole-4-carboxylic acid (229 mg, 1.78 mmol), HATU(681 mg, 1.78 mmol) and DIPEA (0.3 mL, 1.79 mmol) at 0° C., and stirredat rt for 16 h. The reaction mixture was diluted with water (10 mL), thesolid filtered and dried in vacuo to obtain compound 52 (150 mg, 39%) asa brown solid; ¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.14 (s, 1H), 8.06-8.01(m, 2H), 7.35 (d, J=9.0 Hz, 1H), 7.14 (s, 2H), 6.96 (m, 1H), 6.81-6.77(m, 1H), 3.31-3.17 (m, 12H), 1.36 (s, 9H); LCMS (m/z): 529/531 [M+H]⁺.

2-Amino-N-(5-(4-(2-aminoethylsulfonyl) piperazin-1-yl)-2-chlorophenyl)oxazole-4-carboxamide (53)

To a solution of tert-butyl2-(4-(3-(2-aminooxazole-4-carboxamido)-4-chlorophenyl)piperazin-1-ylsulfonyl) ethylcarbamate 52 (150 mg, 0.28 mmol) in DCM (3mL) was added TFA (1 mL) at 0° C., and then the reaction mixture wasstirred at rt for 16 h. The mixture was concentrated in vacuo, basifiedwith saturated NaHCO₃ (aq) (5 mL) at 0° C. and stirred for 10 minutes.The resultant solid was filtered, washed with water, MeOH (2 mL),n-pentane (5 mL), and dried in vacuo to obtain 53 (35 mg, 29%) as an offwhite solid; ¹H NMR (300 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.06 (s, 1H),8.01 (d, J=3.0 Hz, 1H) 7.35 (d, J=8.7 Hz, 1H), 7.14 (s, 2H), 6.81-6.77(m, 1H), 3.31 (m, 4H), 3.23 (d, J=5.4 Hz, 4H), 3.14 (t, J=6.6 Hz, 2H),2.92 (t, J=6.6 Hz, 2H); LCMS (m/z): 429/431 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[2-(dimethylamino)ethyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(54)

2-Amino-N-{2-bromo-5-[4-(ethenylsulfonyl)piperazin-1-yl]phenyl}-1,3-oxazole-4-carboxamide16 (60 mg, 0.13 mmol) and 2M dimethylamine in THF (1 mL) were combinedand irradiated at 100° C. for 30 minutes and then at 120° C. for 30minutes in a Biotage microwave reactor. Further 2M dimethylamine in THF(0.5 mL) was added and the reaction irradiated at 120° C. for 30minutes. The reaction mixture was concentrated in vacuo, and thenpurified by prep. HPLC to obtain compound 54 (5 mg, 8%) as a whitesolid; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.07 (s, 1H), 7.99(d, J=2.7 Hz, 1H), 7.49 (d, J=8.9 Hz, 1H), 7.15 (s, 2H), 6.75 (dd,J=8.9, 2.7 Hz, 1H), 3.34-3.31 (m, 6H), 3.27-3.20 (m, 4H), 2.65-2.60 (m,2H), 2.16 (s, 6H); LCMS (m/z): 501/503 [M+H]⁺.

The following examples were prepared from intermediates 16 or 17 usingan analogous procedure:

2-Amino-N-[2-bromo-5-(4-{[2-(methylamino)ethyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(55)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=3.2 Hz, 1H), 7.49 (d, J=8.9 Hz, 1H), 7.15 (s, 2H), 6.75 (dd, J=8.9,3.2 Hz, 1H), 3.32-3.29 (m, 5H), 3.27-3.18 (m, 6H), 2.86-2.81 (m, 2H),2.27 (s, 3H); LCMS: (m/z)=487/489 [M+H]⁺.

2-Amino-N-[2-chloro-5-(4-{[2-(methylamino)ethyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(56)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=2.7 Hz, 1H), 7.36 (d, J=8.7 Hz, 1H), 7.16 (s, 2H), 6.80 (dd, J=8.7,2.7 Hz, 1H), 3.34-3.30 (m, 5H), 3.26-3.16 (m, 6H), 2.84 (t, J=6.9 Hz,2H), 2.27 (s, 3H); LCMS (m/z): 443/445 [M+H]⁺.

2-Amino-N-[2-chloro-5-(4-{[2-(dimethylamino)ethyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(57)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=2.7 Hz, 1H), 7.35 (d, 0.1=8.9 Hz, 1H), 7.16 (s, 2H), 6.80 (dd, J=8.9,2.7 Hz, 1H), 3.35-3.29 (m, 6H), 3.28-3.18 (m, 4H), 2.68-2.56 (m, 2H),2.17 (s, 6H); LCMS (m/z): 457/459 [M+H]⁺.

2-Amino-N-[2-chloro-5-(4-{[2-(cyclopropylamino)ethyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(58)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=2.7 Hz, 1H), 7.36 (d, J=9.2 Hz, 1H), 7.16 (s, 2H), 6.80 (dd, J=9.2,2.7 Hz, 1H), 3.36-3.27 (m, 4H), 3.26-3.19 (m, 6H), 3.00-2.90 (m, 2H),2.39 (br s, 1H), 2.19-2.01 (m, 1H), 0.36 (dd, J=6.9, 4.6 Hz, 2H), 0.21(dt, J=6.9, 4.6 Hz, 2H); LCMS (m/z): 469/471 [M+H]⁺.

2-Amino-N-[2-chloro-5-(4-{[2-(4-methylpiperazin-1-yl)ethyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(59)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.02 (d,J=2.7 Hz, 1H), 7.36 (d, J=8.7 Hz, 1H), 7.16 (s, 2H), 6.79 (dd, J=8.7,2.7 Hz, 1H), 3.36-3.28 (m, 6H), 3.27-3.24 (m, 4H), 2.65 (t, J=7.1 Hz,2H), 2.46-2.34 (m, 4H), 2.31-2.18 (m, 4H), 2.09 (s, 3H); LCMS (m/z):512/514 [M+H]⁺.

2-Amino-N-(2-chloro-5-(4-{[(2-{[2-(dimethylamino)ethyl]amino}ethyl)sulfonyl]piperazin-1-yl}phenyl)-1,3-oxazole-4-carboxamide(60)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=2.7 Hz, 1H), 7.36 (d, J=8.7 Hz, 1H), 7.16 (s, 2H), 6.79 (dd, J=8.7,2.7 Hz, 1H), 3.37-3.27 (m, 5H), 3.26-3.19 (m, 6H), 2.92-2.87 (m, 2H),2.59-2.55 (m, 2H), 2.28-2.24 (m, 2H), 2.11 (s, 6H); LCMS (m/z): 500/502[M+H]⁺.

2-Amino-N-[2-chloro-5-(4-{[2-(propan-2-ylamino)ethyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(61)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=2.7 Hz, 1H), 7.36 (d, J=8.9 Hz, 1H), 7.16 (s, 2H), 6.79 (dd, J=8.9,2.7 Hz, 1H), 3.34-3.30 (m, 4H), 3.26-3.16 (m, 6H), 2.90-2.84 (m, 2H),2.77-2.65 (m, 1H), 1.69 (br s, 1H), 0.95 (d, J=6.4 Hz, 6H); LCMS (m/z):471/473 [M+H]⁺.

2-Amino-N-{2-chloro-5-[4-({2-[(1-methylpiperidin-4-yl)amino]ethyl}sulfonyl)piperazin-1-yl]phenyl}-1,3-oxazole-4-carboxamide(62)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=2.7 Hz, 1H), 7.36 (d, J=8.9 Hz, 1H), 7.16 (s, 2H), 6.79 (dd, J=8.9,2.7 Hz, 1H), 3.39-3.28 (m, 4H), 3.28-3.15 (m, 6H), 2.94-2.83 (m, 2H)2.74-2.60 (m, 2H), 2.41-2.25 (m, 1H), 2.11 (s, 3H), 1.92-1.62 (m, 5H),1.33-1.00 (m, 2H); LCMS (m/z): 526/528 [M+H]⁺.

2-Amino-N-[2-bromo-6-(4-{[2-(propan-2-ylamino)ethyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(63)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=3.2 Hz, 1H), 7.49 (d, J=8.9 Hz, 1H), 7.15 (s, 2H), 6.75 (dd, J=8.9,3.2 Hz, 1H), 3.34-3.30 (m, 4H), 3.26-3.17 (m, 6H), 2.88 (t, J=6.9 Hz,2H), 2.75-2.67 (m, 1H), 1.80 (br s, 1H), 0.95 (d, J=6.0 Hz, 6H); LCMS(m/z): 515/517 [M+H]⁺.

2-Amino-N-{2-bromo-5-[4-({2-[(1-methylpiperidin-4-yl)amino]ethyl}sulfonyl)piperazin-1-yl]phenyl}-1,3-oxazole-4-carboxamide(64)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=2.7 Hz, 1H), 7.48 (d, J=8.9 Hz, 1H), 7.15 (s, 2H), 6.74 (dd, J=8.9,2.7 Hz, 1H), 3.37-3.27 (m, 4H), 3.27-3.16 (m, 6H), 2.89 (t, J=6.9 Hz,2H), 2.70-2.61 (m, 2H), 2.38-2.30 (m, 1H), 2.11 (s, 3H), 1.95-1.77 (m,3H), 1.77-1.66 (m, 2H), 1.24-1.13 (m, 2H); LCMS (m/z): 570/572 [M+H]⁺.

2-Amino-N-[2-chloro-5-(4-{[2-(ethylamino)ethyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(65)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=2.7 Hz, 1H), 7.36 (d, J=9.2 Hz, 1H), 7.16 (s, 2H), 6.80 (dd, J=9.2,2.7 Hz, 1H), 3.34-3.31 (m, 4H), 3.26-3.17 (m, 6H), 2.88 (t, J=7.1 Hz,2H), 2.52 (q, J=7.1 Hz, 2H), 1.82 (br s, 1H), 0.98 (t, J=7.1 Hz, 3H);LCMS (m/z): 457/459 [M+H]⁺.

2-Amino-N-[2-chloro-5-(4-{[2-(propylamino)ethyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(66)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=2.7 Hz, 1H), 7.36 (d, J=9.2 Hz, 1H), 7.16 (s, 2H), 6.79 (dd, J=9.2,2.7 Hz, 1H), 3.38-3.29 (m, 4H), 3.27-3.16 (m, 6H), 2.87 (t, J=6.9 Hz,2H), 2.49-2.42 (m, 3H), 1.45-1.32 (m, 2H), 0.85 (t, J=7.6 Hz, 3H); LCMS(m/z): 471/473 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[3-(pyrrolidin-1-yl)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(67)

2-Amino-N-(2-bromo-5-{4-[(3-chloropropyl)sulfonyl]piperazin-1-yl}phenyl)-1,3-oxazole-4-carboxamide13 (90 mg, 0.18 mmol), sodium iodide (27 mg, 0.18 mmol) and pyrrolidine(44.5 μL, 0.33 mmol) were dissolved in THF (2 mL). The reaction mixturewas irradiated at 140° C. for 45 minutes in a Biotage microwave reactor.The mixture was concentrated in vacuo and then purified by preparativeHPLC (high pH buffer) to give the product 67 as a white solid (12 mg,13%); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.07 (s, 1H), 7.99(d, J=3.2 Hz, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.16 (s, 2H), 6.75 (dd,J=9.2, 3.2 Hz, 1H), 3.31 (m, 6H), 3.24 (m, 6H), 3.18-3.11 (m, 4H),1.95-1.85 (m, 2H), 1.82-1.66 (m, 4H); LCMS: (m/z)=541/543 [M+H]⁺.

The following examples were prepared from intermediates 13, 14 or 15using analogous procedures:

2-Amino-N-[2-chloro-5-(4-{[3-(dimethylamino)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(68)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=3.0 Hz, 1H), 7.36 (d, J=9.2 Hz, 1H), 7.17 (s, 2H), 6.80 (dd, J=9.2,3.0 Hz, 1H), 3.33-3.28 (m, 4H), 3.26-3.20 (m, 2H), 3.12-3.07 (m, 2H),2.38-2.31 (m, 2H), 2.15 (s, 6H), 1.85-1.78 (m, 2H); LCMS: (m/z)=471/473[M+H]⁺.

2-Amino-N-(2-bromo-5-(4-(3-(dimethylamino)propylsulfonyl)piperazin-1-yl)phenyl)oxazole-4-carboxamide(69)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.07 (s, 1H), 8.03 (s, 1H), 7.95 (d,J=2.8 Hz, 1H), 7.45 (d, J=9.2 Hz, 1H), 7.11 (s, 2H), 6.71 (dd, J=8.9,3.0 Hz, 1H), 3.28-3.23 (m, 7H), 3.23-3.15 (m, 2H), 3.07-3.01 (m, 2H),2.26 (t, J=6.9 Hz, 2H), 2.10 (s, 6H), 1.80-1.72 (m, 2H); LCMS:(m/z)=515/517 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[3-(ethylamino)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(70)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (br s, 1H), 8.07 (s, 1H), 7.99 (d,J=2.7 Hz, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.16 (s, 2H), 6.75 (dd, J=9.2,2.7 Hz, 1H), 3.34 (br s, 1H), 3.34-3.27 (m, 4H), 3.26-3.21 (m, 4H),3.16-3.08 (m, 2H), 2.57 (t, J=6.6 Hz, 2H), 2.54-2.46 (m, 2H), 1.91-1.67(m, 2H), 0.99 (t, J=7.1 Hz, 3H); LCMS (m/z): 515/517 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[2-methyl-3-(pyrrolidin-1-yl)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(71)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=3.0 Hz, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.16 (s, 2H), 6.75 (dd, J=9.2,3.0 Hz, 1H), 3.30-3.22 (m, 10H), 2.86 (br s, 2H), 2.47-2.35 (m, 3H),2.29-2.06 (m, 2H), 1.69 (br s, 4H), 1.13-1.05 (m, 3H); LCMS (m/z):555/557 [M+H]⁺.

2-Amino-N-[2-chloro-5-(4-{[3-(propan-2-ylamino)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(72)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=3.0 Hz, 1H), 7.36 (d, J=8.9 Hz, 1H), 7.16 (s, 2H), 6.80 (dd, J=8.9,3.0 Hz, 1H), 3.30 (s, 4H), 3.26-3.20 (m, 4H), 3.17-3.09 (m, 2H),2.67-2.61 (m, 1H), 2.57 (t, J=6.9 Hz, 2H), 1.81-1.74 (m, 2H), 1.56 (brs, 1H), 0.94 (d, J=6.0 Hz, 6H); LCMS (m/z): 485/487 [M+H]⁺.

2-Amino-N-[2-chloro-5-(4-{[3-(methylamino)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(73)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=3.0 Hz, 1H), 7.36 (d, J=8.9 Hz, 1H), 7.16 (s, 2H), 6.80 (dd, J=8.9,3.0 Hz, 1H), 3.44-3.27 (m, 5H), 3.27-3.20 (m, 4H), 3.19-3.08 (m, 2H),2.61 (t, J=6.9 Hz, 2H), 2.29 (s, 3H), 1.90-1.79 (m, 2H); LCMS (m/z):457/459 [M+H]⁺.

2-Amino-N-[2-chloro-6-(4-{[3-(ethylamino)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(74)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.01 (d,J=3.0 Hz, 1H), 7.36 (d, J=8.9 Hz, 1H), 7.16 (s, 2H), 6.80 (dd, J=8.9,3.0 Hz, 1H), 3.34-3.27 (m, 5H), 3.27-3.20 (m, 4H), 3.16-3.08 (m, 2H),2.57 (t, J=6.6 Hz, 2H), 2.53-2.46 (m, 2H), 1.88-1.69 (m, 2H), 0.98 (t,J=7.1 Hz, 3H); LCMS (m/z): 471/473 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[3-(methylamino)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(75)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (br s, 1H), 8.07 (s, 1H), 7.99 (d,J=2.7 Hz, 1H), 7.49 (d, J=8.7 Hz, 1H), 7.15 (s, 2H), 6.75 (dd, J=8.9,2.7 Hz, 1H), 3.41-3.26 (m, 5H), 3.23 (d, J=3.7 Hz, 4H), 3.15-3.06 (m,2H), 2.54 (t, J=6.6 Hz, 2H), 2.24 (s, 3H), 1.83-1.76 (m, 2H); LCMS(m/z): 501/503 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[3-(propan-2-ylamino)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(76)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=2.7 Hz, 1H), 7.49 (d, J=8.7 Hz, 1H), 7.15 (s, 2H), 6.75 (dd, J=8.7,2.7 Hz, 1H), 3.33-3.27 (m, 5H), 3.27-3.21 (m, 4H), 3.15-3.09 (m, 2H),2.69-2.62 (m, 1H), 2.57 (t, J=6.9 Hz, 2H), 1.81-1.74 (m, 2H), 0.99-0.90(m, 6H); LCMS (m/z): 5291531 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[3-(diethylamino)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(77)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=2.8 Hz, 1H), 7.49 (d, J=8.7 Hz, 1H), 7.16 (br s, 2H), 6.75 (dd, J=9.2,3.2 Hz, 1H), 3.33-3.29 (m, 4H), 3.25-3.22 (m, 4H), 3.13-3.09 (m, 2H),2.61-2.38 (m, 6H), 1.80 (br s, 2H), 0.97 (br s, 6H); LCMS (m/z): 543/545[M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[2-methyl-3-(methylamino)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(78)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=3.0 Hz, 1H), 7.49 (d, J=8.9 Hz, 1H), 7.16 (s, 2H), 6.75 (dd, J=8.9,3.0 Hz, 1H), 3.43-3.19 (m, 10H), 2.85-2.74 (m, 1H), 2.46-2.39 (m, 2H),2.26 (s, 3H), 2.20-2.07 (m, 1H), 1.05 (d, J=6.9 Hz, 3H); LCMS (m/z):515/517 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[3-(ethylamino)-2-methylpropyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(79)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.07 (s, 1H), 7.98 (d,J=2.7 Hz, 1H), 7.48 (d, J=8.8 Hz, 1H), 7.15 (s, 2H), 6.74 (dd, J=8.8,2.7 Hz, 1H), 3.37-3.20 (m, 12H), 2.79 (dd, J=13.7, 8.7 Hz, 1H),2.56-2.42 (m, 2H), 2.14-2.03 (m, 1H), 1.04 (d, J=6.9 Hz, 3H), 0.99 (t,J=7.1 Hz, 3H); LCMS (m/z): 529/531 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[2-methyl-3-(propan-2-ylamino)propyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(80)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=3.2 Hz, 1H), 7.49 (d, J=8.7 Hz, 1H), 7.16 (s, 2H), 6.75 (dd, J=8.7,3.2 Hz, 1H), 3.32-3.22 (m, 10H), 2.80 (dd, J=14.2, 8.7 Hz, 1H),2.71-2.59 (m, 1H), 2.49-2.38 (m, 2H), 2.10-1.99 (m, 1H), 1.05 (d, J=6.9Hz, 3H), 0.98-0.93 (m, 6H); LCMS (m/z): 543/545 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[3-(diethylamino)-2-methylpropyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(81)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=3.2 Hz, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.16 (s, 2H), 6.75 (dd, J=9.2,3.2 Hz, 1H), 3.32-3.21 (m, 11H), 2.80 (dd, J=14.0, 8.9 Hz, 1H),2.49-2.36 (m, 2H), 2.28-2.17 (m, 2H), 2.12-2.04 (m, 1H), 1.04 (d, J=6.4Hz, 3H), 0.93 (t, J=7.1 Hz, 6H); LCMS (m/z): 557/559 [M+H]⁺.

2-Amino-N-[2-bromo-5-(4-{[3-(dimethylamino)-2-methylpropyl]sulfonyl}piperazin-1-yl)phenyl]-1,3-oxazole-4-carboxamide(82)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (s, 1H), 8.07 (s, 1H), 7.99 (d,J=2.7 Hz, 1H), 7.49 (d, J=8.7 Hz, 1H), 7.16 (s, 2H), 6.75 (dd, J=8.7,2.7 Hz, 1H), 3.30-3.20 (m, 10H), 2.89-2.73 (m, 1H), 2.29-1.92 (m, 8H),1.05 (d, J=6.0 Hz, 3H); LCMS (m/z): 529/531 [M+H]⁺.

tert-Butyl-4-{[4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-bromophenyl)piperazin-1-yl]sulfonyl}piperidine-1-carboxylate(83)

To a solution of intermediate 6 (500 mg, 0.55 mmol) in DMF (10 mL) at 0°C. was added TEA (0.57 mL, 1.64 mmol) and tert-butyl4-(chlorosulfonyl)piperidine-1-carboxylate (359 mg, 0.55 mmol) and thereaction allowed to warm to rt and stirred for 2 h. It was then quenchedwith water, diluted with EtOAc and LiCl solution added. The organiclayer was separated and evaporated in vacuo to give 83 (588 mg, 70%) asa yellow powder; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.07 (s,1H), 7.99 (d, J=2.7 Hz, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.15 (s, 2H), 6.74(dd, J=9.2, 2.7 Hz, 1H), 4.05-3.97 (m, 2H), 3.49-3.42 (m, 1H), 3.42-3.36(m, 4H), 3.22-3.16 (m, 4H), 2.84-2.68 (m, 2H), 2.00-1.94 (m, 2H),1.49-1.40 (m, 2H), 1.37 (8, 9H); LCMS (m/z): 613/615 [M+H]⁺.

2-Amino-N-{2-bromo-6-[4-(piperidin-4-ylsulfonyl)piperazin-1-yl]phenyl}-1,3-oxazole-4-carboxamide(84)

To a solution of tert-butyl4-{[4-(3-{[(2-amino-1,3-oxazol-4-yl)carbonyl]amino}-4-bromophenyl)piperazin-1-yl]sulfonyl}piperidine-1-carboxylate83 (0.58 g, 0.95 mmol) in DCM (5 mL) was added TFA (5 mL) at rt and thereaction stirred for 2 h. The reaction mixture was concentrated invacuo, and then saturated NaHCO₃ (aq) solution was added to the residue.The resulting precipitate was filtered to obtain compound 84 (0.37 g,76%) as a solid; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.06 (s,1H), 7.98 (d, J=2.7 Hz, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.15 (s, 2H), 6.74(dd, J=9.2, 2.7 Hz, 1H), 3.58-3.25 (m, 6H), 3.29-3.10 (m, 6H), 2.73-2.63(m, 2H), 2.03-1.94 (m, 2H), 1.68-1.56 (m, 2H); LCMS (m/z): 513/515[M+H]⁺.

2-Amino-N-(2-bromo-5-{4-[(1-methylpiperidin-4-yl)sulfonyl]piperazin-1-yl}phenyl)-1,3-oxazole-4-carboxamide(85)

AcOH (17 μL, 0.29 mmol), formaldehyde (10 μL, 0.13 mmol) and sodiumtriacetoxyborohydride (61 mg, 0.29 mmol) were added to 84 (75 mg, 0.15mmol) in DMF (1 mL) and stirred at rt for 18 h. The reaction wasquenched with saturated NaHCO₃ (aq) solution and extracted with DCM. Theorganics were dried, and then purified by preparative HPLC to obtain 85(12 mg, 16%) as a white solid; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s,1H), 8.07 (s, 1H), 7.98 (d, J=3.0 Hz, 1H), 7.49 (d, J=8.9 Hz, 1H), 7.15(s, 2H), 6.74 (dd, J=8.9, 3.0 Hz, 1H), 3.43-3.35 (m, 4H), 3.21-3.17 (m,4H), 3.16-3.09 (m, 1H), 2.90-2.75 (m, 2H), 2.14 (s, 3H), 1.98-1.78 (m,4H), 1.69-1.52 (m, 2H); LCMS (m/z): 527/529 [M+H]⁺.

The following examples were prepared using analogous procedures:

2-Amino-N-{2-chloro-5-[4-(piperidin-4-ylsulfonyl)piperazin-1-yl]phenyl}-1,3-oxazole-4-carboxamide(86)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.00 (d,J=3.0 Hz, 1H), 7.36 (d, J=8.7 Hz, 1H), 7.16 (s, 2H), 6.79 (dd, J=8.7,3.0 Hz, 1H), 3.43-3.36 (m, 5H), 3.31-3.21 (m, 1H), 3.20-3.15 (m, 4H),3.00-2.95 (m, 2H), 2.48-2.39 (m, 2H), 1.88-1.83 (m, 2H), 1.51-1.44 (m,2H); LCMS (m/z): 469/471 [M+H]⁺.

2-Amino-N-{2-chloro-8-(4-[(1-methylpiperidin-4-yl)sulfonyl]piperazin-1-yl}phenyl)-1,3-oxazole-4-carboxamide(87)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.15 (s, 1H), 8.07 (s, 1H), 8.00 (d,J=3.2 Hz, 1H), 7.36 (d, J=9.2 Hz, 1H), 7.16 (s, 2H), 6.79 (dd, J=9.2,3.2 Hz, 1H), 3.42-3.36 (m, 4H), 3.27-3.11 (m, 5H), 3.02-2.82 (m, 2H),2.20 (br s, 3H), 2.09-1.86 (m, 4H), 1.70-1.58 (m, 2H); LCMS (m/z):483/485 [M+H]⁺.

2-Amino-N-{2-bromo-5-[4-(pyrrolidin-3-ylsulfonyl)piperazin-1-yl]phenyl}-1,3-oxazole-4-carboxamide(88)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.09 (s, 1H), 8.06 (s, 1H), 7.99 (d,J=2.7 Hz, 1H), 7.49 (d, J=9.2 Hz, 1H), 7.16 (s, 2H), 6.74 (dd, J=9.2,2.7 Hz, 1H), 3.78-3.67 (m, 1H), 3.40-3.28 (m, 4H), 3.25-3.15 (m, 5H),3.10 (dd, J=11.9, 8.2 Hz, 1H), 2.96 (dd, J=11.9, 6.4 Hz, 1H), 2.85-2.69(m, 2H), 2.07-1.97 (m, 1H), 1.93-1.84 (m, 1H); LCMS (m/z): 499/501[M+H]⁺.

2-Amino-N-(2-bromo-5-{4-[(1-methylpyrrolidin-3-yl)sulfonyl]piperazin-1yl}phenyl)-1,3-oxazole-4-carboxamide (89)

¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.10 (s, 1H), 8.07 (s, 1H), 7.98 (d,J=3.0 Hz, 1H), 7.48 (d, J=8.9 Hz, 1H), 7.16 (s, 2H), 6.74 (dd, J=8.9,3.0 Hz, 1H), 3.96-3.88 (m, 1H), 3.36-3.33 (m, 4H), 3.21-3.16 (m, 4H),2.85-2.80 (m, 1H), 2.63 (dd, J=10.1, 6.4 Hz, 1H), 2.53-2.46 (m, 2H),2.24 (s, 3H), 2.16-2.08 (m, 1H), 2.06-1.98 (m, 1H); LCMS (m/z): 513/515[M+H]⁺.

PAICS 2D Cell Proliferation Assay

Cell Culture

Frozen cryovial stocks of LM2 and MDA-MB-231 breast cancer cell lineswere thawed and cultured in 4.5 g/L glucose DMEM (31966-047, Invitrogen)supplemented with 10% Fetal Bovine Serum (10500-064, Invitrogen) untilconfluent in a T175 flask. Cells were incubated at 37° C. with 5% CO₂ ina humidified incubator.

Day 1—Cell Plating

Cells were collected from T175 flasks by washing with PBS (10010-056,Invitrogen) and dissociated using Accutase (A6964, Sigma Aldrich). Thiswas followed by centrifuging (1200 rpm, 5 min) and resuspending thecells in 10 ml of media containing 1% penicillin/streptomycin(15140-122, Invitrogen). Cells were counted by trypan blue exclusionmethod using an automated cell counter (Cellometer) and diluted to aconcentration of 40,000/ml. 1000 cells were then seeded into 384-wellwhite-walled plates (Corning, 3707) in a volume of 25 μl. Plates wereIncubated overnight at 37° C. with 5% CO₂.

Day 2—Compound Addition

After 24 hours, PAICS compounds were added onto the assay plates usingan HP D300 Digital Dispenser (Hewlett Packard). 10 mM stocks were usedand dispensed as a 10 point concentration response curve (CRC) ½ logdilution series with a final top concentration of 100 μM. Controls wereadded onto each plate: positive controls included Staurosporine CRC (100μM stock dispensed as a 10 point CRC ½ log dilution series with a finaltop concentration of 1 μM) and PAICS compound MRT00211919 (10 mM stockdispensed as a 10 point CRC ½ log dilution series with a final topconcentration of 100 μM); a high control of Staurosporine (0.1 μM (LM2),0.316 μM (MDA-MB-231) FAC) and a low control of media containing 1%DMSO. A final concentration of 1% DMSO was then added across the platesfor normalisation. Following compound addition, assay plates wereincubated for 72 hours at 37° C. with 5% CO₂.

Day 5—Cytotoxicity and Cell Viability Assays

After 72 h incubation, dead and viable cells were measured in each assayplate. Cytotoxicity was assessed through fluorescent DNA staining usingthe CellTox™ Green Cytotoxicity Assay kit (G8743, Promega) following themanufacturer's multiplexing protocol. Impaired cell membrane integrityresults in the access of dye to stain DNA allowing quantification ofdead cells. Cell viability was determined following ATP quantificationusing the CellTiter-Glo® Luminescent Cell Viability Assay kit (G7572,Promega) following the manufacturer's protocol. ATP released from lysedcells allowed for the generation of a signal proportional to the numberof cells present in the well. All endpoint reads were performed usingthe PheraSTAR Plus (BMG Labtech). Data is expressed as % viable cellsand % dead cells against controls (mean±SEM of duplicate curves).

Biochemical Assay to Measure PAICS Activity

Assay Principle:

The biochemical assay was used to measure PAICS SAICARsynthetase-mediated conversion of CAIR, aspartic acid and ATP to SAICAR,ADP and inorganic phosphate (P_(i)), post-AIR/CAIR equilibration (seeFIG. 3). This was achieved by detecting the ADP generated during thereaction, using Bellbrook Lab's Transcreener ADP² FI assay. An increasein the fluorescence intensity is directly proportional to the amount ofPAICS activity.

The Transcreener ADP² FI assay is a homogeneous competitive displacementfluorescence intensity assay which uses direct immunodetection of ADP.Displacement of the tracer by ADP causes an increase in the fluorescenceat excitation 590 nm and emission 617 nm (FIG. 3).

Method:

5 μl WT full length PAICS (final assay concentration, fac, 2.5 nM) inbasic buffer, added to black, non-binding, 384-well plates (Corning#3575), columns 1 to 22. 5 μl basic buffer was added to columns 23+24(negative control). PAICS stock at 31.9 μM from the PI's lab (SteveFirestine). Basic buffer contained 50 mM Tris-HCl pH8 and 0.5 mM EDTA,fac.

1 μl compound in 100% DMSO added per well, or 1 μl 100% DMSO to positivecontrols (columns 1+2) and negative controls (columns 23+24). Finalassay top concentration of compound either 1 mM or 30-μM, seriallydiluted with half-log dilutions across the plate in duplicate (one10-point concentration response curve across wells 3 to 12, anotheracross 13 to 22). Compounds pre-incubated with PAICS enzyme for 30 minsat RT. 2 μl CAIR added (fac 10 μM) in basic buffer plus 25 mM MgCl₂ and50 mM KHCO₃ fac to all wells. CAIR stock 50 mM from Steve Firestine'slab. 1 hr RT incubation for AIR-CAIR equilibration. NB: ˜50% CAIR isdecarboxylated during equilibration therefore 5 μM remains for thesynthetase reaction.

2 μl ATP/aspartic acid added (fac 30 μM/180 μM) in reaction buffer toall wells. 30 mins RT incubation for appropriate level of ATP turnover.Reaction buffer contained basic buffer plus 10 mM DTT, 0.01% BSA and0.01% Brij 35, fac.

10 μl ADP detection reagent added to all wells (as per instructions,Transcreener ADP² FI kit, BellBrook Labs #3013-10K). Incubation for 1 hat RT to allow antibody equilibration. Fluorescence intensity determinedusing a Tecan Safire2 (excitation at 590 nm, emission at 617 nm).

The results of the above assays for selected compounds of the inventionare shown in Table 1.

PAICS Biochemical Activity IC₅₀

A=<10 nM; B=10-25 nM; C=25-100 nM; D=>100 nM; nt: not tested

PAICS 2D Cell Proliferation Activity EC₅₀

A=<100 nM; B=100-250 nM; C=250-500 nM; D=500-1000 nM

TABLE 1 PAICS PAICS 2D Cell Example Biochemical Proliferation numberIC₅₀ EC₅₀ 19 D D 20 B D 21 B B 22 D B 23 B B 24 B B 25 B B 26 nt D 27 ntD 28 B C 29 C D 30 D D 31 C D 32 C D 33 C D 34 C D 35 C D 44 B D 45 B C46 D D 53 C D 54 A B 55 A A 56 B C 57 B D 58 C D 59 B C 60 B C 61 B B 62B C 63 A A 64 A B 65 B B 66 B B 67 A A 68 B C 69 A A 70 A A 71 nt A 72 AB 73 B C 74 A C 75 A B 76 A A 77 A A 78 nt A 79 nt A 80 nt A 81 nt B 82nt B 84 A B 85 A B 86 B C 87 B C 88 nt B 89 nt BCRISPR Editing

Cas9 nuclease-mediated gene editing (Sander and Joung, 2014) wasperformed using a CRISPR RNA guide sequence (TACGAATTGTTAGACAGTCC, PAM:AGG) targeting exon 3 in the human PAICS gene (accession number:NM_006452).

Stable cell clones were isolated and gene disruption confirmed by DNAsequencing and the absence of PAICS cellular protein expressionconfirmed by Western blotting.

A small set of compounds were tested against wild type LM2 and CRISPRedited LM2 in the 2D cell proliferation assay to establish to whatextent the cellular potency was driven by PAICS mediated effects. Thedata is shown in Table 2: compounds tested had no effect on CRISPRedited cells, confirming the on-targets effect as being PAICS mediated.

TABLE 2 Potency shift: CRISPR Cell Example EC₅₀/WT number Cell EC₅₀23 >500 24 >500 55 >500 69 >500Evaluation of Compound 69 in MDA-MB-231-Dlux Xenograft Model

Protocol:

Mice were implanted with MDA-MB-231 Dlux cells in the mammary fat pad.Mice were treated with test agent, (Compound 69), standard of carecontrol agent, docetaxel, or vehicle control, once tumours reached amean volume of ˜100-200 mm³. Tumours were measured every 2-3 days usingelectronic calipers and tumour volume estimated using the formula 0.5(L×W²). Treatment continued for 2 weeks. Mice were observed and bodyweight measured daily

Mice:

MF-1 (HsdOla:MF1-Foxn1^(nu))

Female 5-8 weeks

n=12 per group

Cells:

MDA-MB-231 Dlux (Bioluminescent MDA-MD-231 variant)

Implantation Site: Mammary fat pad (1:1 matrgel/PBS)

Groups:

Vehicle (10% DMSO)

Compound 69 (Oral 25 mg/kg twice daily)

Docetaxel (IV 5 mg/kg twice weekly)

Results

Mice treated with PAICS inhibitor (25 mg/kg bid) showed a statisticallysignificant reduction in growth of primary tumour. The results are shownin FIG. 4.

Evaluation of the Growth of PAICS CRISPR KD MDA231 Cells inChorioallantoic Membrane (CAM) Model

Protocol:

Fertilised white leghorn eggs were incubated at 37.5° C. with relativehumidity for 9 days. At E9 the chiorioallantoic membrane (CAM) wasdropped by drilling a small hole through the eggshell into the air sacand a cm² window was cut in the eggshell above the CAM. 2×10⁶ MDA231cells were added onto the CAM of the egg (groups included: wild typeMDA231 cells; CRISPR KD control cells; PAICS CRISPR KD clone 3 cells;and PAICS CRISPR KD clone 14 cells). At E18 the upper portion of theCAM, with the tumour, was removed, transferred in PFA 4%. After 48 hourin PFA, tumour was washed three times in PBS and the tumour wascarefully cut away from normal CAM tissue. Tumour was then weighted anda one-way ANOVA analysis done on data for the 4 groups Tun and PAICSCRISPR KD (KD clones 3 & 14), as well as wild type MDA231 cells andcontrol CRISPR cells. Table 3 shows the groups for the study.

TABLE 3 Name group Amount of Nb of eggs Group description in the reportcells/egg grafted/group Group 1 Wild type WT 2.10⁶ 21 MDA231 cells Group2 Modified MDA231 Ctrl 1 2.10⁶ 21 cells-Control Group 3 Modified MDA231KD 3 2.10⁶ 21 cells-KD 3 modification Group 4 Modified MDA231 KD 142.10⁶ 21 cells-KD 14 modification

Results

Table 4 presents the mean value, SD, SEM and p-value of tumour weight(mg) for each experimental group at E18 (n is the number of eggs aliveat the end of the study). The mean tumour weight (mg) measured in thedifferent experimental groups is shown in FIG. 5.

TABLE 4 Tumor p value p value p value weight vs vs. vs. n (mg) SD SEM WTCtrl 1 KD3 WT 13 133,492 38,420 10,656 / / / Ctrl 1 19 104,433 17,3833,988   0.0068  / / KD 3 17 37,435 6,246 1,515 <0.00001 <0.00001 / KD 1415 35,195 8,181 2,112 <0.00001 <0.00001 0.3878

PAICS CRISPR KD clones (KD3 and KD14) of MDA231 modified cells showed asignificant reduction in tumour development compared to wild type andcontrol modified MDA231 cells. See FIG. 6.

Various modifications and variations of the described aspects of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes of carrying out the invention which are obvious tothose skilled in the relevant fields are intended to be within the scopeof the following claims.

The invention claimed is:
 1. A compound of formula (I), or apharmaceutically acceptable salt or ester thereof,

wherein: B is a saturated or partially unsaturated monocyclic orbicyclic, heterocyclic group optionally substituted by one or more R¹⁰groups and optionally containing one or more CO groups; X is selectedfrom SO₂, CO₂, CO, CONR¹¹, NR¹¹CO and (CR¹²R¹³)_(p); R¹ is independentlyselected from halogen, OR¹⁴, SR¹⁴ and R¹⁴; R² is selected from H andalkyl; R³ is selected from H, alkyl, cycloalkyl and heterocycloalkyl,each of which is optionally substituted by one or more substituentsselected from NR²⁴R²⁵ and R²⁶, or R³ is linked to one of R⁴, R⁵, R⁶, R⁷,R⁸ or R⁹ to form a saturated heterocyclic group; or R² and R³ are linkedtogether with the nitrogen to which they are attached to form asaturated heterocyclic group optionally containing one or moreadditional heteroatoms selected from O, N and S, and optionallysubstituted by one or more R²⁷ groups; each R⁴ and R⁵ is independentlyselected from H, alkyl, (CH₂)_(s)OR¹⁵ and (CH₂)_(t)NR¹⁶R¹⁷; or one of R⁴and R⁵ is H or alkyl and the other is linked to R³ to form a saturatedheterocyclic group, or one of R⁴ and R⁵ is linked to one of R⁸ or R⁹ toform a cyclic group; each R⁶ and R⁷ is independently selected from H,alkyl, (CH₂)_(u)OR¹⁸ and (CH₂)_(v)NR¹⁹R²⁰; or one of R⁶ and R⁷ is H oralkyl and the other is linked to R³ to form a saturated heterocyclicgroup; each R⁸ and R⁹ is independently selected from H, alkyl,(CH₂)_(w)OR²¹ and (CH₂)_(x)NR²²R²³; or one of R⁸ and R⁹ is H or alkyland the other is linked to R³ to form a saturated heterocyclic group; orone of R⁸ and R⁹ is linked to one of R⁴ and R⁵ to form a cyclic group;R¹⁰ is selected from alkyl, OH, halogen, alkoxy, CO₂-alkyl, COOH,CO-alkyl and CN; R¹¹, R¹², R¹³, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²²,R²³, R²⁴ and R²⁵ are each independently H or alkyl; R¹⁴, R²⁶ and R²⁷ areeach independently alkyl; m, q and r are each independently 0, 1 or 2,such that the sum of m+q+r is 2, 3 or 4; p is an integer selected from0, 1 and 2; and s, t, u, v, w and x are each independently 0, 1, 2, 3 or4.
 2. A compound according to claim 1 wherein B is a monocyclic 5- or6-membered saturated or partially unsaturated heterocyclic groupoptionally substituted by one or more R¹⁰ groups.
 3. A compoundaccording to claim 1 wherein: each R⁴ and R⁵ is independently selectedfrom H and alkyl; or one of R⁴ and R⁵ is H or alkyl and the other islinked to R³ to form a saturated heterocyclic group; each R⁶ and R⁷ isindependently selected from H and alkyl; or one of R⁶ and R⁷ is H oralkyl and the other is linked to R³ to form a saturated heterocyclicgroup; and each R⁸ and R⁹ is independently selected from H and alkyl; orone of R⁸ and R⁹ is H or alkyl and the other is linked to R³ to form asaturated heterocyclic group.
 4. A compound according to claim 1 whichis of formula (Ib), or a pharmaceutically acceptable salt or esterthereof,

wherein Y and Z are both N, or one of Y and Z is N, and the other is CH;and R¹⁻⁹, X, m, q and r are as defined in claim
 1. 5. A compoundaccording to claim 4 wherein Y and Z are both N.
 6. A compound accordingto claim 1 wherein R¹ is selected from Br, I, Cl, OMe, SMe and Me.
 7. Acompound according to claim 1 wherein X is CO₂ or SO₂.
 8. A compoundaccording to claim 1 wherein: R² is selected from H, methyl, ethyl andisopropyl; and R³ is selected from methyl, ethyl, isopropyl andcyclopropyl.
 9. A compound according to claim 1 wherein R² and R³ arelinked together with the nitrogen to which they are attached to form a5- or 6-membered saturated heterocyclic group.
 10. A compound accordingto claim 1 wherein: m is 1 or 2; q is 1 or 2; r is 0; R⁸ and R⁹ are eachindependently H or alkyl; and R⁶ and R⁷ are each independently H oralkyl.
 11. A compound according to claim 1 wherein: m is 1; q is 1 or 2;r is 0; one of R⁸ and R⁹ is H or alkyl and the other is linked to R³ toform a saturated heterocyclic group; and each R⁶ and R⁷ is independentlyH or alkyl.
 12. A compound according to claim 1 wherein: m is 1; q is 1;r is 1 or 2; R⁸ and R⁹ are each independently H or alkyl; one of R⁶ andR⁷ is H or alkyl and the other is linked to R³ to form a saturatedheterocyclic group; and R⁴ and R⁵ are each independently H or alkyl. 13.A compound according to claim 1 which is selected from the following,

and pharmaceutically acceptable salts and esters thereof.
 14. Apharmaceutical composition comprising a compound according to claim 1and a pharmaceutically acceptable carrier, diluent or excipient.
 15. Amethod of treating a disease alleviated by inhibition of PAICS in asubject in need thereof wherein the disease is a proliferative disorder,said method comprising administering to the subject a therapeuticallyeffective amount of a compound according to claim
 1. 16. A method oftreating a disease alleviated by inhibition of PAICS in a subject inneed thereof wherein the disease is metastatic cancer, said methodcomprising administering to the subject a therapeutically effectiveamount of a compound according to claim
 1. 17. A method of treating adisease alleviated by inhibition of PAICS in a subject in need thereofwherein the disease is cancer or leukemia, wherein the method comprisesadministering to the subject a therapeutically effective amount of acompound according to claim
 1. 18. A method of treating a diseasealleviated by inhibition of PAICS in a subject in need thereof whereinthe disease is selected from breast cancer, colon cancer, prostatemelanoma, bladder, pancreatic, head and neck and ovarian cancer, with orwithout metastasis, haematological cancer, acute myeloid leukemia (AML),chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL),multiple myeloma (MM) and non-Hodgkins lymphoma, wherein the methodcomprises administering to the subject a therapeutically effectiveamount of a compound according to claim
 1. 19. A method of treating adisease alleviated by inhibition of PAICS in a subject in need thereofwherein the disease is breast cancer, wherein the method comprisesadministering to the subject a therapeutically effective amount of acompound according to claim 1.